PFF1320C an Essential but Presumptive Plasmodium Myosin Light Chain

Melody Robinson Wright

March 2017

A Dissertation Presented to the Faculty of Drexel University College of Medicine In partial fulfillment of the Requirements for the Degree of Doctor of Philosophy

DEDICATION

This dissertation is dedicated to my grandmother, Dorothy Ann Gilmore Robinson and to my parents Colonel Ronald Robinson and Reverend Mary C. Sloan.

iii

ACKNOWLEDGEMENTS

The completion of this dissertation would not have been possible without

the unwavering support of my family, friends, and many within the Drexel

Community. Throughout this scientific journey I have been blessed with many

amazing mentors. I would like to thank Dr. Lawrence W. Bergman for allowing

me the pleasure to learn and grow in his lab over the past seven years. His

strong passion for science and overall enthusiasm for life created the perfect lab

environment for which I was able to discover confidence in myself as an

independent scientist. His support for my project has always been steadfast even

at times when I begin to question it all. For this I am truly grateful.

To the remaining members of my dissertation committee Dr.

James Burns, Dr. Theodore Taraschi, Dr. Anand Mehta, and Dr. Patrick Loll I

appreciate all your support, helpful suggestions, advice, and constant motivation

through the more troubling times of this endeavor. I would especially like to

thank Dr. Burns his help with troubleshooting experiments. More importantly I

would like to thank him to taking the time out to meaningfully ask me how I was

doing. Simple gestures such as this can go a long way in the life of graduate

student.

To great lab environment is only as great as the people who are in it. To

Tom Daly, words could never express my gratitude for everything you’ve taught iv

me as well as your contribution to my research. You taught me how to look at

things differently and appreciate the beauty in the techniques in ways that I will

never forget. To Dr. Bethany Jenkins, thank you for helping me transition into the

lab, for being an amazing lab mate, and more importantly a great friend. It was a

pleasure to grow into a scientist along side you. Thank you to Aaron Tocker for

his assistance with different aspects of the project. I would like to also thank Dr.

Sumit Kumar for helping me in the early days of starting my project.

I would like to thank the Vaidya lab for all their collaborations and

support throughout the years. I would like to give a special thanks to Joann

Morrisey, for her wisdom and helpful advice when it came to culturing parasites.

I would like to thank all colleagues within the Micro/Immuno department

along with my batch mates for making my time here at Drexel memorable, all

the collaborative talk and friendship. Thank you to Andrea Partridge, Dr. Ming

Yang, Dr. April Pershing, Liz Parzych, Sezin Patel, Lindsay Kleinwaks, and

Jacqueline Schneider.

To my parents, sisters, grandmothers, and extended family. Thank you so

much for your enormous support and for being my biggest cheerleaders. I could

not have made it to the end without you. To my husband Marcus Wright, thank

you for being a solid rock while these crazy winds were blowing. To my dogs

Jada and Jujubee, thank you for always giving me a reason to smile whenever I

came home. I love you all!

Table of Contents

Chapter 1 ...... 1

Introduction ...... 1

History Of Myosins ...... 6

Actin And Unique Actin Binding Proteins ...... 10

Apicomplexan Myosins ...... 12

History Of Myosin Light Chains ...... 20

Conditional Knockdown Strategies ...... 34

Proximity Dependent Labeling ...... 38

Thesis Aims...... 39

CHAPTER 2 ...... 41

MATERIALS AND METHODS ...... 41

Plasmodium Cell Culture And Transfection ...... 42

Western blot ...... 42

Microscopy ...... 43

Generation of parasite expression vectors ...... 43

Genomic DNA Isolation From Parasite Pellet ...... 46 vi

Growth Assay For Flow Cytometry ...... 47

Isolation Of Biotinylated Proteins ...... 47

ATc Treatment ...... 48

Mass Spectrometry ...... 48

CHAPTER 3 ...... 51

RESULTS ...... 51

Potential Essential Nature Of PFF1320c ...... 52

Generation Of Epitope Tagged Transgenic PFF1320c Parasites And Expression In The Blood

Stages...... 52

Cellular Distribution Of PFF1320c ...... 57

PFF1320c Co-Localize With PfMyoC In a Inconsistent Manner...... 58

Knockdown Of PFF1320c Does Not Show An Effect On Growth Of The Parasite...... 59

The TetR-Dozi Aptamer System Enhances Regulation of PFF1320C...... 63

Biotinylation In 1320-BirA* Parasites Is Distinct From Cytoplasmic BirA* And Localizes To The

Cytoplasm...... 69

The Identification Of Presumed Myosin Complex Proteins Through Mass Spectrometry of

MyoB-BirA* and MTIPB-BirA* Pulldowns...... 75

CHAPTER 4 ...... 97

DICUSSION/CONCLUSION ...... 97

REFERENCES ...... 111

APPENDENCIES...... 120

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LIST OF TABLES

Table 1.1 Apicomplexan myosins naming convention...... 14

Table 1.2 Grouping of Apicomplexan myosin sequences...... 19

Table 3.1 Proteins identified by mass spectrometry in 1320-BirA* samples...... 85

Table 3.2 PfMyoC proteins found in biotinylation...... 88

Table 3.3 Proteins found similar in data sets from 1320-BirA*-Aptamer...... 90

Table 3.4 PfMyoB and PfMTIPB associated proteins identified by mass

spectrometry...... 94

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LIST OF FIGURES

Figure 1.1 The Plasmodium Life Cycle...... 4

Figure 1.2 Myosin Class XIV Phylogeny ...... 17

Figure 1.3 Repertoire of myosin light chain-like proteins in Apicomplexa...... 31

Figure 1.4 Amino Acid Sequence Alignment of Plasmodium Myosin Light Chains (EF-

Hands)...... 32

Figure 1.5 PFF1320c conserved only in Primate Plasmodia...... 33

Figure 3.1 Generation of transgenic PFF1320c using the PLN System...... 53

Figure 3.2 Succesfful expression of tagged PFF1320c in blood stage parasite ...... 54

Figure 3.3 PFF1320c is essential for blood stage growth...... 55

Figure 3.4 PFF1320c is expressed during the trophozoite stage in the blood stage of

the parasite...... 60

Figure 3.5 PFF1320c is localized to the cytosole of the parasite...... 61

Figure 3.6 Co-immunofluorescence of PFF1320c and PfMyoC...... 62

Figure 3.7 Schematic of the glmS ribozyme reverse genetic tool...... 65

Figure 3.8 PFF1320c is knocked down in the presence of Glucosamine...... 66 ix

Figure 3.9 Successful knockdown of PFF1320 in the 1320-HA-glms KO transgenic

parasite line upon addition of glucosamine substrate...... 67

Figure 3.10 Growth of transgenic parasites are not effected by glucosamine mediated

knockdown...... 68

Figure 3.11 Conditional knockdown with TetR-Dozi aptamer...... 70

Figure 3.12 The aptamer system regulates translation of 1320-HA-Aptamer transgenic

parasites...... 71

Figure 3.13 Confirmation of aptamer induced regulation of translation in 1320-HA-

Aptamer transgenic parasites...... 72

Figure 3.14 Expression and localization of 1320-Bir*-HA...... 76

Figure 3.15 1320-BirA* exhibits a unique biotinylation profile...... 77

Figure 3.16 1320-BirA* biotinylation is localized to the cytosol...... 78

Figure 3.17 1320-HA-BirA* Biotinylation ...... 79

Figure 3.18 The small molecule ATc stringently controls the expression of 1320-BirA*-

Aptamer...... 83

Figure 3.19 Biotinylation in 1320-BirA*-Aptamer parasites...... 84

Figure 3.20 Proteins identified in parasites expressing BirA* fusion proteins...... 87

Figure 3.21 Proteins from 1320-BirA*-Aptamer pulldowns identified by mass

spectometry...... 90

Figure 3.22 Biotinylated proteins co-localize with MyoB and MTIPB BirA* fusions. .... 93

Figure 3.23 Biotinylation of MyoB and MTIPB BirA* fusion...... 94

Figure 3.24 Proteins from MyoB and MTIPB BirA* fusion pull downs ...... 96 x

TABLE OF ABBREVIATIONS

AA- Amino Acid

ADP – Adenosine Diphosphate

ADF- Actin-depolymerizing Factor

Arf- ADP-ribosylation factor

ARP- Actin related protein

ATc- Anydrotetracycline

ATP - Adenosine Triphosphate

ATS1- alpha-tubulin suppressor 1

C- carboxy

CaM- Calmodulin

DD – destabilization domain

DDD- DFHR degradation domain

DHFR- dihydrofolate reductase

DOZI- development of zygote inhibited

ER- endoplasmic reticulum xi

F-Actin- Filamentous actin

FERM- band four-point-one, exrin, radixin, moesin domain

FV- Food Vacuole

GAP- glideosome associated protein

GAPM- glidesome-associated protein membrane spans

HRS- hours

HA- hemagluttinin

HV- hemoglobin vacuole

IMC- inner membrane complex

JAS- jasplakinolide

KD- Knock Down

KO- Knock Out

MCM – mini chromosome maintenance

MHC- myosin heavy chain

MLC- myosin light chain

MTIP- myson tail interacting protein

MTIPB- myosin tail interacting protein B

MyoA- Myosin A

MyoB- Myosin B

N- amino

PCR- polymerase chain reaction

PH- pleckstrin homology xii

PI- post invasion

PIP2- phosphatidylinsotil 4-5

PM- plasma membrane

PPM- parasite plasma membrane

PVM- parasitophrous vacuolar membrane

PV- parasitophorous vacuole

RBC- red blood cell

RNAi- RNA interference

Shld-1- shield 1

SHV- small hemoglobin vacuole

TJ- tight junction

TMP- trimethorprim

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ABSTRACT

PFF1320C AN ESSENTIAL BUT PRESUMPTIVE MYOSIN LIGHT CHAIN

Melody R. Wright

Lawrence W. Bergman

Little is known regarding the nature and function of the myosin motors of Plasmodium.

Analysis of the genome reveals that there are six myosin heavy chains but only two,

PfMyoA and PfMyoB, have been characterized with the role of PfMyoA, along with its cognate light chain partner MTIP, being directly involved in the process of parasite invasion. PFF1320c is annotated in the Plasmodium genome as a putative myosin light chain but to date there is no biochemical evidence to support this designation.

Interestingly, this molecule is only found in the primate malaria parasites and is not found in the rodent parasites. Preliminary studies of PFF1320c have shown that the protein is essential due to our inability to delete the gene. Expression of a tagged version of the gene at an ectopic site permitted the successful knockout of PFF1320c. xiv

Studies are currently underway to identify the myosin heavy chain (MHC) partner and other potential binding partners using PFF1320c fused to promiscuous biotin ligase

(BirA*) to label proteins in close proximity to the presumptive light chain, where the

1320-BirA* fusion is also subject to translational regulation by use of a small molecule aptamer. We are also working to phenotypically elucidate the role of this presumptive actin-myosin motor within the parasite using the glmS ribozyme or the aptamer system as an alternate knockdown strategy. Further characterization of this presumptive myosin light chain and identification of the MHC partner will provide an insight into the role of this unique myosin motor in the growth and development of the parasite.

Chapter 1

Introduction

Malaria is an infectious disease that has killed more humans than any other disease and nearly half of the world’s population is at risk of contracting the disease. In

2015 there were approximately 212 million cases and an estimated 429,000 deaths [1].

Though increased prevention and control measures have led to a 60% reduction in malaria rates globally since 2000, this disease burden is still disproportionately high in

Sub-Saharan Africa, a region home to 89% of malaria cases and 91% of malarial deaths

[1]. Five species of Plasmodium can infect humans under natural conditions:

Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium knowlesi and Plasmodium malariae. Of these five, P. falciparum is the most prevalent and deadliest, as it is responsible for most malaria related deaths globally. Transmission of the Plasmodium parasite depends on an intermediate insect vector for transmission to occur. Anopholine mosquitoes, during a blood meal, inject sporozoites into the skin, which after crossing the endothelial barrier enter the circulation to reach the liver; an organ that is critical for the next phase of the parasite life cycle, the pre-erythrocytic stage [2-4]. Once in the liver sinusoids, the sporozoites encounter the endothelial barrier and pass through various liver cells before actively invading hepatocytes.

Sporozoites divide mitotically into thousands of liver merozoites, which on release invade erythrocytes, thus starting the asexual blood stage lifecycle of the parasite. In P. falciparum the erythrocytic stage is a 48-hour cycle during which parasites mature through three different stages, ring, trophozoite, and schizonts. During this cycle the parasites replicate to form 16 to 32 daughter merozoites that are released during egress. The egress and reinvasion of the merozoites also marks the manifestation of 3 clinical symptoms that are characteristic of the disease such as fever, chill, muscle ache, and headache. Some of the merozoite-infected blood cells leave the cycle of asexual replication. Instead, merozoites in these cells develop into sexual forms of the parasite, male and female gametocytes. These, when taken up during a blood meal by the mosquito, develop into mature sexual forms called gametes. Male and female gametes fuse to form diploid zygotes. The zygote develops to actively moving invasive ookinetes that traverses the mosquito midgut wall and transforms into an oocyst from which sporozoites are released. These sporozoites then migrate to the mosquito’s salivary glands for injection into the human host during the next blood meal. Plasmodium generates three invasive stages, sporozoites, merozoites, and ookinetes. Although the morphology of the blood stage merozoites differs slightly, merozoites share some characteristics like polarized morphology and apical organelles with other invasive forms of Plasmodium as well as other apicomplexan parasites (Figure 1.1). Among the many vital functions of an obligate intracellular parasite, host cell invasion is a perequisite for survival and replication. Most invasion stages are highly motile and can move rapidly on solid substrates (1-10 uM sec -1), but have no apparent specialized structures such as cilia, flagella, or pseudopods for locomotion [5]. Unlike sporozoites, merozoites do not show an apparent gliding movement [6]. The invasion of a red blood cell by a merozoite is an essential multistep process [7]. 4

Figure 1.1: The Plasmodium life cycle. The life cycle of Plasmodium species that infect mammals. Symptoms of malaria are caused by cycles of parasite multiplication inside erythrocytes. One cycle is initiated as a result of erythrocyte invasion by a merozoite form that develops into a ring, then trophozoite, and finally a schizont, generating new merozoites. A cycle typically lasts ~ 48 hours (hrs). Some intraerythrocytic parasites transform into male or female gametocytes, which are taken up by a mosquito. Gametocytes egress from erythrocytes, activate into gametes and fuse in the mosquito midgut lumen. The motile zygote, called an ookinete, crosses the gut epithelium to transform into an oocyst, in which thousands of sporozoites develop. Sporozoites are released into the mosquito body cavity and later pass through salivary gland cells to enter the salivary ducts. After transmission into the skin of the mammalian host during a bite by the mosquito, motile sporozoites reach the liver, where they invade hepatocytes. One intra-hepatocytic sporozoite generates tens of thousands of hepatic merozoites, which re-enter the bloodstream and invade erythrocytes. Republished with permission from [8].

Initial contact between the merozoites and erythrocyte is a crucial step, as the parasite must distinguish between erythrocytes that are competent for invasion and other cell types. Initially, the merozoites undergo random, reversible attachments on the red blood cell surface [9] . The parasite then reorients itself to bring the apical end in prominence with the host cell. It has been shown that the tight interactions between the parasite receptors and ligand on the red blood cell (RBC) membrane can be uncoupled from subsequent steps by pretreating parasites with the actin depolymerizing agent cytochalasin B. These findings were first shown in P. knowlesi [10] and later confirmed in P. falciparum [11].

The commitment of merozoites to invade is marked by the formation of a junction between the merozoites and the RBC as well as the coordinated induction of the parasitophorous vacuole (PV) [12]. The tight junction (TJ) develops between the apical end of the invading parasite and the target host. The coordinated secretion of micronemes and rhoptries is a very sophisticated feature of apicomplexan invasion.

Micronemes are the first organelles to discharge proteins thought to participate in gliding and host cell recognition [13] followed by the release from rhoptries. The contents of the rhoptries contribute to the invasion process by providing material for the formation of the parasitophorous vacuole as well as modifying the host cell prior to and post invasion [14-16]. The essential microneme protein (AMA1) binds exposed regions of the rhoptry protein RON2 resulting in the close interaction between the parasite and host cell [12, 17]. As invasion proceeds, the TJ transforms into a circumferential ring that traverses across the merozoites surface. The composition of 6 the tight junction is made up of both micronemal (AMA1) and rhoptry (RON2, RON4, and RON5) proteins. Microneme secretion is regulated by an increase in cytoplasmic calcium release from parasite intracellular store before host cell interaction, whereas rhoptry secretion is dependent on the contact of the parasite with the host cells. An actomyosin motor, to be described in detail below, powers the parasite invasion process.

History of Myosins

Myosins constitute a large superfamily of proteins that share a common domain, which has been shown to interact with actin, hydrolyze adenosine triphosphate (ATP), and produce movement [18, 19]. Myosins are molecular motors that diversified very early during eukaryotic evolution [20, 21] and are involved in diverse cellular functions like muscle contraction, cell locomotion, cytokinesis, vesicle transport, Golgi organization and sensory transduction. Most eukaryotes rely on myosins and only a few taxonomic groups like red algae and diplomonad protists appear to live without them

[22]. There are up to 40 different myosin genes within mammals and genetic mutations within myosins are linked to serious pathologies like myopathies, blindness and hearing loss.

Myosins heavy chains (MHC) are comprised of three structural subdomains consisting of the head/motor, neck and tail. A myosin can either be single or double headed. The conserved head domain or motor domain interacts with actin and binds

ATP. The neck domain, also known as the lever arm, possesses a consensus IQ motif, involved in binding myosin light chains or calmodulin. The tail domains are the most 7 diverse domains and vary widely in length and in sequence. The tail domains of many myosins contain coiled-coil forming sequences that allow the molecules to dimerize and produce two-headed molecules. Tail domains can also contain functional motifs such as

SRC homology 3 (SH3) domains, GTPase activating (GAP) domains, band four-point-one, exrin, radixin, moesin (FERM) domains, and pleckstrin homology (PH) domains [18]. A large majority of myosins and myosin classes adhere to the TEDS rules where they have either the amino acid (AA) T,E,D, or S at a position 16 residues upstream of conserved sequence element DALAK [23] in the head region. Enzymatic activities of these myosins, are dependent upon phosphorylation at the TEDS site. For some myosins phosphorylation at TEDS site residues is required for regulatory functions such as actin- activation of ATPase activity, growth, actin organization, and endocytosis [24] .

The myosin motor is powered through use of the energy from ATP hydrolysis, which generates force or directed movement along actin filaments. In the absence of

ATP, the cycle begins with the myosin head tightly bound to an actin filament in a rigor state. [25]. This change affects the neck region of the myosin that binds the light chains, which acts as a lever arm to displace the myosin head about 5 nm. The products of ATP hydrolysis (adenosine diphosphate (ADP) and Pi) remain bound to the myosin head in a

“cocked” position. The myosin head then rebinds at a new position on the actin filament, resulting in the release of ADP and Pi triggering the “power stroke,” in which the myosin head regains its original conformation and restarts the cycle. All characterized myosin motors move towards the barbed (+) end of actin filaments with the exception of myosin VI, an unconventional myosin that moves toward the pointed 8

(-) end of actin. It achieves its reverse-direction movement by rotating its lever arm in the opposite direction to the conventional myosin lever arm movement [26]. This backwards movement is attributed to a 50 AA insertion prior to the IQ motif that could potentially alter the convertor subdomain [18].

Phylogenetic analysis of the conserved motor domains groups myosins into 35 classes of which 13 are present in humans [27]. Class II myosins were discovered over

70 years ago and since it was the only class known for decades, traditionally it was considered ‘conventional’ and other classes found afterward became known as

‘unconventional’. Members of this class are found in muscles and cytoplasm of animals.

This class also constitutes the skeletal muscle myosin, which powers F-actin sliding in sarcomeres during muscle contraction [28]. Class II myosins are composed of two heavy chains and two pairs of light chains. Dimerization of the heavy chain tail gives rise to the double-headed phenotype. The amino-terminal portion of the heavy chains contains the prototypical motor domain and two IQ motifs in the neck. Like many classes of myosins this class has been sub-classified into 4 groups based on its motor domain sequences.

The sarcomeric myosins from striated and cardiac muscles fall into one group whereas vertebrate smooth and non-muscle myosins fall into another. Myosins from lower eukaryotic species make up the third subclass while myosin from fungi comprises the fourth [18] . Class I myosins were next discovered and the subsequent classes were numbered in the order of the discovery of the founding member of the class. The number of myosin genes present in mammals is conservatively estimated at 40 from 9 classes I, II, III, VI, VII, IX, X and XV [29] [18]. All eukaryotic animal cells usually have at least one myosin II and multiple myosin I genes [18].

Class XIV myosins are restricted to apicomplexan parasites and other members of the Alveolata. Most myosins categorized under class XIV in apicomplexans all share the consensus actin binding and ATP-hydrolyzing sites within their head regions, their necks lack a putative IQ motif or contain degenerate IQ motifs, and in some cases their tails are shorter as compared to other known MHC. PfMyoE, grouped under class XIVb, does not share this phenotypical truncated tail as seen with PfMyoA and PfMyoB [29,

30], which fall into the same class. The Plasmodium myosins will be discussed in greater detail below.

Class VI myosins exhibit unusual directionality with movement towards the minus end of actin filaments [26]. The presence of two unique inserts near the nucleotide binding domain and between the myosin head domain and predicted IQ motif of 40-50 AA is thought to be responsible, for this phenomenon [31].

Class XXII myosins have been implicated in diverse functions like signal transduction and transcriptional regulation due to their formation of β–propeller structures formed by the four to six WD40 repeats [29]. Sequences from apicomplexans within this class are predicted to feature an extended neck domain with multiple IQ motifs which is a characteristic feature of myosins of class V and related classes VIII, XI, and XIIII which are often positioned in close proximity to class XXII in tree topologies

[32]. 10

Actin and Unique Actin Binding Proteins

Motility plays a key role in the ability of Plasmodium parasites to cause infection, by enabling them to penetrate deep into human tissues. In eukaryotes, actin and other proteins that regulate actin activity primarily drive cell motility. Actin is a major protein constituent of the cytoskeleton and plays a key role in several cellular processes such as cell shape regulation, cell motility, intracellular trafficking and cytokinesis [33, 34].

Biochemically actin exists in both monomeric or globular (G-actin), or polymeric or filamentous (F-actin) forms and the distribution is not affected by actin-polymerizing agents like phallodin, MgCl2, exogenous actin, spermine or phosphatidylinositol-4,5 bisphosphate (PIP2). Apicomplexan actin exists primarily in a monomeric form consistent with the fact that the apicomplexan actin both in vivo and in vitro only forms very short unstable filaments [35-40]. The ratio of globular to filamentous actin reflects an organism’s activity level as the highly motile nature of T. gondii tachyzoites reflects the high content of globular actin. Despite its central role in apicomplexan motility, under normal conditions actin filaments are undetectable by electron microscopy [39, 41].

Several studies have used jasplakinolide (JAS), an actin filament-stabilizing drug to visualize cytoplasmic actin filaments towards the apical end of the parasite [39, 40, 42].

Treatment with JAS also shows that tight regulation of actin polymerization appears to be the rate limiting step in apicomplexan gliding motility [35].

Plasmodium spp. express two isoforms of actin with Actin I (PfACT1) being the major isoform. PfACT1 is expressed throughout the life cycle of the parasites. It shares

~83% sequence identity with actins from yeast and vertebrate muscle [43]. The minor 11 isoform actin II (PfACT2) is present only in the gametocytes and mosquito stages including sporozoites [43, 44] and contains a single intron. Actin I is an essential component of parasite motor machinery responsible for the unique gliding motility and host cell invasion by the parasites, [10, 40] while actin II is only required in male gametogenesis [45]. The two gene sequences bear 79% homology to each other. Actin in T. gondii (TgACT1) is encoded by a single copy gene containing one intron and shares maximum identity to PfACT1 at 93% and C. parvum actin at 88%. All three contain the form of actin having an additional acidic residue at the amino (N) terminus relative to conventional vertebrate actins [35].

Until recently, it was proposed that a continuous filamentous actin scaffold up to a length of 10 μm would be necessary to power parasite gliding motility. However,

PfACT1 has been shown to assemble in short filaments in vivo [38]. This supports the notion that since the motor complex has a reverse orientation, actin filaments should be short. Immobilized motor myosins are anchored to the inner membrane complex (IMC) and serve as the scaffold [46]. Short actin polymers are pulled backwards by a set of myosin motors and passed on to the next motor unit. Hence, Plasmodium actin no longer needs to form typical microfilaments that determine shape and orientation of a cell but oligomerize into small units that serve as the backbone for a limited number of receptor-ligand complexes [38].

The repertoire of actin-binding and actin-regulation proteins in the apicomplexan genome is lacking as compared to that of other eukaryotes [47].

Apicomplexans lack the actin-related protein complex Arp2/3, one of the main 12 machineries orchestrating actin nucleation in eukaryotes [48, 49]. However there are sequences within the Plasmodium genome annotated as putative Arps. Homologs of conserved actin-associated proteins; coronin and actin-depolymerizing factor (ADF) have been characterized in Apicomplexa. A coronin homolog coded by a single copy gene is found in Plasmodium [49]. ADF/cofilins bind both G- and F-actins and catalyze depolymerization of actin filaments into actin monomers. Plasmodium spp. have two isoforms of ADF/cofilins (ADF1 and ADF2). PfADF1 is expressed throughout the parasites life cycle and is essential for gliding movement and parasite invasion. PfADF2 binds both

G- and F- actins, severs actin filaments and in contrast to canonical ADF/cofilins, induces exchange of ADP with ATP on bound actin monomers [50]. PfADF2 is not expressed during the infective erythrocytic stage of malarial parasites [38].

Apicomplexan Myosins

In the absence of locomotive organelles, apicomplexan parasites have developed an unusual mode of substrate dependent gliding locomotion that is necessary for invasion [51]. Inhibitory functions of cytochalasins on invasion by Plasmodium, Eimeria and Toxoplasma on actin filaments has been demonstrated establishing that motility is essential for invasion and depends on the parasite’s own cytoskeleton. Treatment with cytochalasin B blocked P. knowlesi merozoite invasion in red blood cells though the merozoites could still attach to the cell, showing that attachment is uncoupled from invasion [10].

A limited and divergent repertoire of myosins has been identified in two apicomplexans, T. gondii and P. falciparum. Apicomplexan myosins were commonly 13 named in order of discovery. However this caused confusion among non-orthologous sequences being designated with the same letter. To clarify this situation, Foth et al. proposed a systematic naming convention for apicomplexan myosin heavy chains (MHC) in an effort to designate orthologous myosins across all apicomplexans by the same capital letter. This naming system was based on homology to T. gondii myosins as this species contains the largest known myosin repertoire of any apicomplexan for which genome data is available. Consequently, six apicomplexan myosins have been renamed.

MyoC, MyoD, and MyoF of Plasmodium were changed to MyoF, MyoJ, and MyoK, respectively. MyoB and MyoE in the genus Plasmodium are exceptions to this general naming convention as they are not orthologous to MyoB and MyoE found in T. gondii and therefore their name remains unchanged [29] (Table 1.1).

Toxoplasma has the largest repertoire of myosin proteins with 11 known heavy chain sequences belonging to different classes, with 5 being grouped into class XIV

(TgMyoA, B, C, E, and F). In 2002, the role of MyoA in Toxoplasma was elucidated by a conditional known down approach [52]. TgMyoA is associated with the myosin light chain (MLC) MLC1 and plays an essential role in parasite motility and invasion.

TgMyoB/C is encoded by alternative splicing of a single gene differing only in their carboxy (C) -terminus. TgMyoB shows a punctate distribution throughout the cytoplasm while TgMyoC is selectively concentrated at the anterior and posterior polar rings of the inner membrane complex [53]. TgMyoC has also been shown to compensate for the absence of TgMyoA [54]. Over expression of TgMyoB or TgMyoC results in a defect in

Table 1.1: Apicomplexan Myosins Naming Convention *MyoF, *MyoJ, and *MyoK of Plasmodium were previously known as MyoC, MyoD and MyoF, respectively. Toxoplasma gondii (Tg), Plasmodium falciparum (Pf), and Eimeria tenella (Et) Adapted from [29].

15 parasite cell division, replication, and formation of residual bodies suggesting that both these myosins could be involved in daughter cell budding and separation [53]. Of the 11 myosin heavy chains MyoD is the smallest and shares similarities in structure, localization, and kinetic properties with TgMyoA [29, 55]. Unlike TgMyoA, TgMyoD is shown to be dispensable for tachyzoites propagation and motility and is highly expressed in bradyzoites. Coccidian specific MLC2, to be described in further detail, is the MLC binding partner of TgMyoD. TgMyoD mutants lacking the tail fail to localize properly, strengthening an earlier theory of the tail domain being an essential determinant of localization in apicomplexan class XIV myosins [29, 55]. TgMyoF is a class

XXII myosin that is highly conserved across the apicomplexan phylum and localizes to the apicoplast during division. TgMyoH is an indispensable actin-based motor associated with the microtubules of the conoid and plays an indispensable role in initiating the first step of motility, which is subsequently relayed by MyoA to enact effective gliding and invasion [56]. The tail region of MyoH possesses alpha-tubulin suppressor 1 domains

(ATS1) that are essential for the anchoring of this motor to the conoid. Depletion of

TgMyoH dramatically impacts motility, invasion and egress from the infected cells

[56]. Localization and biological function of TgMyoG, TgMyoK , and TgMyoI are unknown at this time.

Six myosins, MyoA, MyoB, MyoC, MyoD, MyoE and MyoF have been identified in the Plasmodium genome. These six myosin may very well represent the full P. falciparum myosin complement but as myosins structurally resemble kinesins with no discernable sequence homology there remains the possibility that highly divergent 16 myosin/s exist that have not been identified by approaches taken to date [57]. Of these three (PfMyoA, PfMyoB, and PfMyoE) cluster under the apicomplexan specific Class XIV myosins. PfMyoC is found under the monophyletic clade of class XXII and MyoD and

MyoF under Class VI. Of the Plasmodium myosins, MyoA is the only unambiguous homolog of other apicomplexan MyoA myosins and plays a critical role in gliding motility and invasion. PfMyoA and its myosin light chain partner MTIP (myosin tail interacting protein) are components of the glideosome. PfMyoB and PfMyoE, which belong exclusively to Plasmodium myosins, do not unambiguously cluster with any apicomplexan sequences [29] (Figure 1.2). PfMyoB is expressed in all invasive stages of the life cycle and the protein is found localized at the extreme apical end of motile and invasive parasites though its biological function is unclear [58]. Translation of PfMyoA protein occurs very late in the erythrocytic cell cycle during merozoites maturation.

Likewise PfMyoB is synthesized very late in schizogeny, and its location in merozoites is distinct from, and anterior to, that of a range of known proteins present in the rhoptries, rhoptry neck or micronemes. Unlike MyoA, MyoB is not associated with the glideosome complex proteins nor is it found in association with MTIP. The mRNA expression of PfMyoE shows that it is absent from ring stages, low in trophozoites, but highly abundant in schizonts. mRNA expression of PfMyoC occurs predominately in trophozoites. PfMyoD is expressed at the earliest in late trophozoites and more strongly in schizonts. PfMyoF is high for all asexual stages and increases steadily throughout the cell cycle [57] The localization and biological function of PfMyoC, PfMyoD, PfMyoE, and

PfMyoF remains indistinct at this time. 17

Figure 1.2: Myosin Class XIV Phylogeny. PfMyoB and PfMyoE belong exclusively to Plasmodium [29].

To identify myosins within the various Apicomplexa, a polymerase chain reaction

(PCR) strategy was previously used to clone the first class XIV myosins from Toxoplasma

[59]. Degenerate PCR primers corresponding to two highly conserved sequences found in most myosin classes (GESGAGKT and EAFGNAKT) were used to amplify sequences from P. falciparum, Neospora caninum, Eimeria tenella, Sarcocystis mris, and

Cryptosporidium parvum. The resulting nucleotide sequences were cloned sequenced and then subject to BLAST search programs to provide comparison with sequences available in the public databanks. Of the species examined, several myosin sequences were successfully amplified and all shared either close or exact identity to previously described myosins from Toxoplasma or Plasmodium or to myosin from other parasites

(Table 1.2). Neospora expresses five different myosins, several with close similarity to class XIV myosins. Eimeria and Sarcocystis revealed five and six myosins respectively.

With the exception of one Eimeria myosin nearly identical to T. gondii MyoB, all showed closest alignment to either TgMyoA or PfMyoA. Seven myosin sequences were obtained from Babesia of which 4 were similar to TgMyoA or PfMyoA (50%-93% identity), one with matching identity to TgMyoA, one to TgMyoB and one showing 60% similarity to

PfMyoC. The six myosin sequence fragments obtained from Cryptosporidium genomic library all showed closest resemblance to TgMyoA or PfMyoA (50-93% identity) including one that was identical to PfMyoA. All myosin sequences found in the various parasites were most closely rated to one of the T. gondii or Plasmodium myosins that have been characterized indicating that diversity in Apicomplexa is in fact limited [60].

Table 1.2: Grouping of apicomplexan myosin sequences. Sequences are grouped according to the similarity to the previously-defined Toxoplasma gondii (Tg) or Plasmodium falciparum (Pf) myosins Nc= Neospora Caninum; Et= Eimeria tanella; Sm= Sarcocystis muris; Bb= Babesia bovis; Cp= Crptospordium parvum [60].

As limited diversity is more the exception than the rule most Apicomplexa myosins are classified under the unconventional class XIV [61], including previously identified

Toxoplasma and Plasmodium myosins. This suggest that Apicomplexa may have evolved and subtly diversified their own unique set of myosins enabling them to accomplish all the cellular functions such as cell motility, intracellular transport, endocytosis and signal transduction that are accomplished by a different and greater diversity of myosin in other cell types [19].

History of Myosin Light Chains

MLC’s are as diverse as the associated heavy chains and have structural and regulatory functions that can mediate the association of an MHC with a binding partner or determine the intracellular localization of an MHC [30]. MLCs are calmodulin (CaM)- related proteins, which contain four EF-hand motifs with helix (E)-loop-helix (F) topology that normally bind calcium, though some have lost this calcium binding property. They associate non-covalently with a 20-25 AA basic amphiphilic helix, called IQ motif(s) that contains the consensus sequence IQXXXRGXXXR located in the MHC neck domain. The number of IQ motifs present in the necks of different myosins can vary between zero and six, and the number of light chains predicted to bind to a single MHC can vary from between one to seventeen [62]. IQ motifs are numbered starting with IQ1 for the motif with closest proximity to the motor domain [30].

Biochemical and structural studies within the conventional Class II myosins revealed that each myosin II heavy chain contained two IQ motifs that associated exclusively with two light chains commonly referred to as essential light chain (ELC) and 21

F chain (RLC) [30]. RLC’s are termed regulatory for their involvement in the regulation of enzymatic activity of some myosin II heavy chains. Upon removal of the ELC from myosin II heavy chains under harsh conditions, enzymatic activity of the holoenzyme is lost which gave rise to the term “essential” [63]. Based on early studies a central dogma was established that conventional myosins bind only one ELC and one RLC [64] however; this specificity is not seen in unconventional myosins. In unconventional myosins, ELC’s and RLC’s exhibit less specificity and can bind together, independently from each other, or in conjunction with CaM and potentially other light chains in various sequential orders to the IQ motifs in unconventional MHCs [30].

With all apicomplexans species, virulence relies on the parasites ability to perform a unique form of cellular locomotion termed “gliding motility.” Gliding motility allows parasites to transverse host tissue and invade as well as gives them the ability to egress host cells. This type of substrate dependent movement is initiated in response to

Ca2+ signaling events and is powered by the force of an actomyosin motor termed the glideosome. The glideosome is a multiprotein complex confined to the space between the PM and the IMC at the parasite’s pellicle. The current model suggests that the glideosome comprises the unique type XIV myosin MyoA [65]. Previous attempts to knock out the TgMyoA gene have failed indicating that the gene is essential, however

Frenal et al. were successfully able to knockout TgMyoA through use of CRISPR/CAS9- mediated gene disruption [54]. Conditional knockdown of the gene using a tetracycline- inducible transactivator system where the gene was knocked out in the presence of a second regulatable copy in tachyzoites [66] showed severe impairment of gliding 22 motility. This impairment compromised the ability of the parasites to invade as well as egress [52]. The light chain partner of TgMyoA, MLC1, was identified and shown to be associated with 53 AA of the C-terminus of the TgMyoA heavy chain [46] and together they play an essential role in gliding, invasion, and motility. The role of binding calcium is not preserved in TgMLC1 as it presents atypical EF-hands where the Ca2+-binding residues are not conserved, suggesting that Ca2+ is not required for binding to the MHC

[64, 67]. ELC1 and ELC2 are essential light chains that together with MLC1 and the MyoA neck region define the lever arm, which consist of the neck region in association with regulatory light chains, essential light chains, or calmodulins (CAM). The common theme of redundancy and plasticity within apicomplexans invasion and motility machinery is displayed in the fact that ELC1 and ELC2 have redundant roles in the

TgMyoA glideosome and their shared role is important for all glideosome dependent processes [54, 68, 69]. Both light chains interact with TgMyoA in a mutually exclusive manner and share the same binding site. Unlike other redundant mechanisms seen within TgMyoA/TgMyoC and GAP45/GAP70/GAP80, ELC1 and ELC2 appear to be associated with the glideosome under physiological condition and not only upon genetic removal of components.

The glideosome is anchored to the IMC by transmembrane glideosome associated proteins (GAPs) GAP40 and GAP50 [65]. TgGAP45 associates with the outer face of the IMC in mature parasites through its N-terminal lipid modifications whereas

TgGAP50, the first membrane protein identified in the glideosome of apicomplexan parasites, is an integral membrane glycoprotein found on the inner membrane complex 23 of both mature and immature parasites [70]. Assembly of the glideosome complex is a two-step process that is also reflected in the subcellular distribution of the different subunits. TgMyoA, TgMLC1, and TgGAP45 are synthesized in the endoplasmic reticulum

(ER). These proteins are only found associated within the inner membrane complex of mature parasites and assemble first into a soluble complex known as the proto- glideosome within the cytoplasm. TgGAP50 is synthesized within the cytoplasm and found in the inner membrane complex of immature daughter parasites as well as mature parasites where it then associates with the complex and becomes anchored in the membrane forming the glideosome proper [70]. It is suggested that the conserved

C-terminal cytoplasmic domain of TgGAP50 most likely associates with the proto- glideosome as interaction is prevented by deletion of this domain [70]. GAP45 is essential for gliding, invasion, and egress. This dual function protein recruits MLC1-

MyoA to the IMC by binding critical residues within the N-terminal extension of MLC1 as well as preserves pellicle integrity during invasion through its N-terminal acylation and coiled coil domain [69]. Final assembly of the glideosome complex is regulated by the phosphorylation of two serine residues on GAP45. Association of the proto-glideosome with GAP50 is prevented until it reaches the IMC, where dephosphorylation enables assembly of the complex. Function is suggested to be conserved throughout apicomplexan parasites for which sequence data is available, orthologues similar to

TgGAP45 and TgGAP50 have been found in other species [70].

GAP70, a coccidian homolog of GAP45, shares several identical features; however, it differs in that its localization is restricted to the apical cap of the IMC of 24 parasites that possess a conoid. GAP70 can partially substitute for GAP45 function, thus confirming its association with the MyoA-MLC1 complex. GAP70’s dispensability implies that its role could either be compensated by GAP45 or it has a more prominent contribution during a different stage [69].

GAP40 is a fifth component of the glideosome that is conserved across the apicomplexan phylum with peak expression during the schizonts stage of P. falciparum.

It is an integral IMC protein analogous to GAP50 that presumably anchors the motor to the IMC.

TgMLC2 was identified as the light chain partner of TgMyoD and is found localized at the pellicle of tachyzoites. Like TgMLC1, TgMLC2 exhibits an N-terminal extension that has proven to be necessary and sufficient to target TgMLC2 to the pellicle as loss of this region causes the MLC2 to remain cytosolic [71]. The C-terminal CaM-like domain of TgMLC2 is necessary and sufficient to interact with MyoD as it substitutes for the function of the tail by bringing the motor to the site of action. As a complex, MyoD-

MLC2 is anchored to the plasma membrane via its N-terminal extension without assistance of any accessory proteins it is believed that three putative palmitoylation sites in its N-terminal extension likely carry this out [71]. In large contrast to GAP50, which is responsible for the immobilization of the MyoA-MLC1 motor to the matrix membrane of the IMC [72], MyoD-MLC2 is fluidly associated with the PM, which most likely reflects a fundamentally distinct motor function. TgMLC2 completely disappears in

MyoD-knock out (KO) parasites similar to the dramatic drop of TgMLC1 observed in

TgMyoA-inducible knockout strains [52]. These losses of MLCs in the absence of their 25

MHC partners suggest that the stability of each component within a myosin motor relies on the interaction between light chain and heavy chain [71]. The MyoD-MLC2 adopts a unique closed and compact conformation in which MLC2 forms a tight clamp around the

MyoD-tail helix. This composition is not observed in the structures of other MHC-MLC complexes making it an attractive target for the development of anti-coccidian drugs

[71].

Toxoplasma possesses at least seven MLCs for which localization was determined by epitope tagging via single homologous recombination at the endogenous locus. TgMLC3, TgMLC5, and TgMLC7 were found to localize exclusively to the conoid

[56]. TgMyoH is associated with TgMLC1, TgMLC5, and TgMLC7. TgMLC5 exclusively associates with TgMyoH at the conoid and is no longer detectable at this location upon depletion of MyoH . Interestingly, upon TgMyoH depletion, TgMLC7 was still present at the conoid though its level of expression decreased after 24h. In contrast to TgMLC1,

TgMLC5 and TgMLC7 are simultaneously dispensable and are not required to sustain or regulate TgMyoH function indicating the possibility that of another MHC, which could be present at this same location [56].

In Plasmodium, MTIP, also known as MLC1, binds to its MHC partner PfMyoA and is the only myosin motor complex in Plasmodium that has been well characterized and its essential role in sporozoites gliding motility and merozoites invasion is well established [46, 73, 74]. MTIP is distantly related to MLCs and functions as a regulatory or essential light chain for PfMyoA. Using the neck and tail domains as bait, the light chain partner of PyMyoA was identified by a yeast two-hybrid screen using a P. yoelii 26 cDNA library for screening [46]. The unusually short PyMyoA tail is formed by the C- terminal 15 AAs and was shown to be crucial for specifically interacting with MTIP.

Mutagenesis of a dibasic motif within the tail domain of MyoA abolished interaction with MTIP. These findings were quite similar to earlier studies where mutagenesis of the dibasic motif in the tail abolished correct localization of TgMyoA to the tachyzoite periphery [75]. Immunoelectron microscopy confirms the presence of MTIP in the IMC of sporozoites. TgMLC1, the MTIP ortholog of Toxoplasma shows this same localization

[76].

The MyoA/MTIP complex is also found expressed in the blood stages of

Plasmodium parasites. Expression of MTIP can be seen 39 hrs after invasion into the 48 hr life cycle with peak expression found at 42-45 hrs after invasion [77].

Immunofluorescence (IFA) localized MTIP to the periphery of merozoites within schizonts, which is similar to the previous localization of MTIP to the IMC complex of sporozoites of P. yoelii [46].

The critical interaction between MTIP and MyoA facilitates the gliding action of the parasite. The structure of MTIP in complex with MyoA was deduced by crystallization of 120 residues of P. knowlesi MTIP with the P. yoelii MyoA C-terminal tail

[73]. The crystal structure reveals that the residues from the non-calcium binding EF- hand region of MTIP form the MyoA binding site, and of the MTIP residues engaged in critical interactions with MyoA, most are all conserved among all Plasmodium species

[78]. When binding to the MyoA tail α helix, MTIP undergoes major conformational change. At neutral pH, P. falciparum MTIP in complex with the MyoA tail adopts a 27

“closed conformation” with two domains of the C-terminal region surrounding the

MyoA tail helix. The P. knowlesi structure obtained at cryogenic temperatures adopts an

“open conformation” with most of the interaction provided by the C-terminal domain.

In both conformations, the interactions of the MyoA helix with the N-terminal domain of MTIP are limited whereas the C-terminal domain provides a deep hydrophobic groove to which the MyoA tail helix binds. Previous studies have shown that the 15 AA long synthetic MyoA peptide is suitable for inhibiting the growth of P. falciparum parasites whereas no effect on parasite growth was seen with a randomized 15-mer or mutant forms of the peptide [73]. Using a hybrid-structure-based method, which is suitable for the discovery of small molecule inhibitors of protein-protein interactions, Kortagere et al., designed eight compounds that belong to a unique pyrazole-urea class. The compounds identified in this studies have shown to be effective antimalarial agents as they inhibit protein-protein interaction in a competitive nature between MTIP and

MyoA that in turn prevents the growth of malarial parasite and alters gliding motility of rodent sporozoites [78].

The MyoA-MTIP motor also plays a role in Rab11A-mediated vesicular trafficking during the formation of IMC and generation of daughter parasites [79]. IFA showed

Rab11A partially colocalizes with the glideosome component GAP45 and pull-down assays confirmed its physical interaction with MTIP. This supports the notion that the

MyoA/MTIP motor drives Rab11A-mediated transport. Ablation of Rab11A function resulted in the loss of vesicle formation and incomplete daughter cell separation. Late components of the glideosome like MLC/MTIP, GAP45 and MyoA failed to integrate with 28 the IMC in daughter parasites expressing a dominant negative form of Rab11A. Based on these results, a proposed model suggested that the Rab11A mediated vesicular transport delivers a new plasma membrane between maturing IMC and also delivers components of the proto-glideosome to the IMC [70, 79].

The glideosome machinery between Toxoplasma and Plasmodium is very similar as the Plasmodium MyoA motor was found to have orthologues of TgMLC1, TgGAP45, and TgGAP50 in blood stages known as PfMTIP, PfGAP45, and PfGAP50 respectively

[74]. Pulse-chase experiments show that the Plasmodium glideosome assembly undergoes a two-step process resembling formation of the Toxoplasma glideosome.

PfGAP50 is translated and inserted into the IMC during its formation while the other members MyoA, MTIP, and PfGAP45 form a separate complex known as the proto- glideosome that is delivered to the IMC of mature parasites. The expression of these proteins reflects the differences in formation with PfGAP50 expression at around 30-32 hrs-post invasion corresponding with development of the IMC. Expression of PfGAP45,

MTIP and MyoA appears at 40-42 hrs-post invasion.

Glideosome-associated protein with multiple membrane spans (GAPMs) is a novel family of apicomplexan specific proteins that constitute a three-member family of six-pass transmembrane proteins that localize to the IMC [80]. Localized to the IMC these proteins form large detergent-resistant complexes of the order of >200 kDa, possibly being the components of intramembranous particles. These proteins have been suggested as strong candidates for performing an IMC-anchoring role as they interact 29 with the cytoskeleton alveolins on one side and to a lesser degree with the actin-myosin motor components, GAP45/50 on the other.

MyoB light chain MLCB/MTIPB has recently been identified as the light chain partner of MyoB and shown to be localized to the apex of merozoites, at a location indistinguishable from MyoB [58]. The inability to knock out MTIPB in P. falciparum and

P. berghei suggest that the protein is essential for parasite survival. MTIPB is the largest myosin light chain identified in any species and is composed of two parts: the EF-hand containing calmodulin-like C-terminal domain and the larger N-terminal domain. The calmodulin-like C-terminal domain is able to bind a peptide from the extreme C- terminus of MyoB. The N-terminal extension of MTIPB is far larger than that of MTIP, which has been demonstrated to be important for its interaction with GAP45 [58] and compromises more than 75% of the MTIPB sequence. Analysis of this N-terminal sequence suggests it forms a dimeric-coiled coil and through its dimerization, this unusual light chain helps PfMyoB achieve functionality and correct localization.

A database search across apicomplexan genomes to find EF-hand containing protein sequences closely related to CaM revealed two MLCs within Plasmodium that are orthologous to Toxoplasma’s MLC4 and MLC7 (Figure 1.3). [71]. One of the light chains identified is PFF1320c, which is a putative myosin light chain annotated in the

Plasmodium database. However, there is no biochemical evidence to verify the actual protein’s role. This protein varies significantly in size to that of the other Plasmodium

MLCs with a total amino acid count of 139 where MTIP and MTIPB have 204 and 652

AAs respectively. While sequence variation is seen at the amino terminal ends of MTIP 30 and MTIPB, PFF1320c appears to lack this extension (Figure 1.4A). However, amino acid alignment of the C-terminal regions of the three molecules shows similarity as well as containing the non-calcium binding EF-hand motif. Both PfMTIP and PfMTIPB show an almost 50% sequence similarity to PFF1320c within this C-terminal region (Figure 1.4B).

The six-myosin heavy chains, MTIP, and MTIP-B are conserved throughout primate- infecting plasmodia (P. falciparum, P vivax, and P. knowlesi) and in the rodent-infecting plasmodia (P. berghei, P. chabaudi, and P. yoelii). Surprisingly, PFF1320C is only found in the primate infecting plasmodia despite synteny existing in the adjacent regions among all the Plasmodium genomes (Figure 1.5). Within Plasmodium only ~85% of genes are orthologous with the remainder being specific to individual Plasmodium species. This represents innovations and changes that have arisen during the evolution and adaptation of these species to their environment and life. Lineage or species-specific genes and multi copy genes have been shown to be integral to host species interactions, and many arise via later transfer or de novo creation [81].

The de novo generation of genes has played an important role in the virulence of apicomplexans [81, 82]. As parasite motility and invasion are critically dependent on an association between MHCs and MLCs, we can deduce from the characterization of myosin motors found within T. gondii that the role these motors play in Plasmodium is equally as essential. The key to learning and understanding more about the intracellular movements of the parasite is through its molecular motors.

Figure 1.3: Repertoire of myosin light chain-like proteins in Apicomplexa. Phylogenetic tree of apicomplexan myosin light chain-like sequences. Arrow indicates PFF1320c. The full species names (in alphabetical order) are as follows: B. bovis, Babesia bovis; C. hominis, Cryptosporidium hominis; C. muris, Cryptosporidium muris; C. parvum, Cryptosporidium parvum; E. tenella, Eimeria tenella; N. caninum, Neospora caninum; P. berghei, Plasmodium berghei; P. falciparum, Plasmodium falciparum; P. knowlesi, Plasmodium knowlesi; P. vivax, Plasmodium vivax; P. yoelii, Plasmodium yoelii; T. annulata, Theileria annulata; T. gondii, Toxoplasma gondii; T. parva, Theileria parva.

Figure 1.4: Amino acid Sequence Alignment of Plasmodium Myosin Light Chains (EF- Hands). A. Sequence alignment of myosin light chains from their C-terminal regions. B. Percent identities and similarities of these regions within PfMTIP, PfMTIPB, and PFF1320c.

Figure 1.5: PFF1320c conserved only in primate plasmodia. PFF1320c is only found in the primate infecting Plasmodia, despite the synteny existing in the adjacent regions among all the Plasmodium genomes.

To gain further insight into the composition and role of myosin motors within P. falciparum identification of MHC binding partners for the putative MLCs and determining a genetic phenotype associated with depletion in Plasmodium are imperative.

Conditional Knockdown Strategies

The study of essential genes in P. falciparum is difficult due to the haploid nature of the organism. Though knockout technology has been established, null mutants cannot be created through conventional knockout due to genes having essential roles in the haploid blood stages of the parasite except in special cases where the gene function can be complemented chemically [83]. This is why new tools that provide improved genetic technologies that allow for the precise, reversible, and temporal manipulation of the Plasmodium genome at several possible levels are essential to study the biology of the parasite. In contrast to knockout for testing loss of gene function, there are additional methods available for regulating gene expression at multiple points, throughout transcription and post-translation stages. Inducible systems control expression at the transcriptional level whereas RNA interference (RNAi) or ribozyme- mediated strategies have their effects posttranscriptonally at the level of mRNA stability

[84]. Through use of RNAi, one of the more commonly used tool in eukaryotes, manipulation of the parasite has been reported but has not shown to be functional as typical RNAi machinery is absent from the parasite genome [85].

To control gene expression at the level of protein stability, a mutated version of the human FKBP12 protein (ddFKBP), known as the destabilization domain (dd), is fused 35 to the N or C-terminus of a protein and dramatically interferes with protein stability, resulting in rapid degradation of the protein by the proteasome [84, 86]. Protein stability in transfected parasites can be modulated by a small FKBP ligand, Shield1

(Shld1), which specifically interacts with the ddFKBP, folds it and blocks degradation [87] allowing for regulation of protein levels in mammalian cells. This system has been demonstrated to be successful for proteins expressed at low concentration and where the maximum difference achievable between ligand-stabilized and destabilized target protein level is about five-fold [88-90]. Limitations of this system include the prohibitive nature of use due to cost of the shld-1 and its association with toxicity in long-term experiments [90], leakiness, as the amount of degradation may not be sufficient to produce a phenotype for some proteins, growth delay as it has been shown that the amount of Shld-1 inhibits growth at 0.5 uM, the concentration needed to stabilize the target protein [90, 91], and lack of function as dd-fused proteins may not function correctly when stabilized by the ligand. This is further supported by the inability to integrate the DD sequence at many different P.falciparum loci [91].

A conceptually similar approach based on the Escherichia coli dihydrofolate reductase (DHFR) enzyme, the DHFR degradation domain (DDD) is of similar efficiency to the DD system and can modulate protein levels whether episomally expressed or integrated into the genome [92]. When fused to a protein of interest, the instability of the DHFR enzyme is conferred to the fused protein resulting in rapid degradation of the entire fusion protein. The DDD is stabilized in a rapid, reversible, dose-dependent manner by an advantageously inexpensive small molecule folate analog trimethoprim 36

(TMP), which is a moderately potent anti-malarial drug [93]. TMP is toxic to parasites therefore, before use, prior genetic modification of the parasite to express a human

DHFR marker is required to make the parasite resistant to TMP. This system suffers many of the problems discussed above, except cost.

Transcription inducible systems such as the Tetracycline-inducible transactivator system have been employed for the generation of inducible conditional mutants within

Plasmodium. While this effort is tedious and work intensive, several groups have successfully applied this technique to study the function of essential genes [94].

However, there are several disadvantages that could prevent the formation and analysis of inducible conditional mutants such as a phenomenom called “squelching” where unwanted toxic effects are created as a result of overexpression of a factor that interacts with the transcription machinery [66, 95]. In contrast, naturally occurring self- cleaving RNAs (ribozymes) are modular elements that retain function in different RNA contexts, and can be engineered to respond to ligands [96]. Ribozymes have been utilized in systems to overcome the need for expression of trans-acting factors since all the information necessary for ectopic gene regulation is contained within the autocatalytic RNA [66, 97]. The glmS ribozyme from gram-positive bacteria is part of the mRNA encoding the essential enzyme that synthesizes glucosamine-6-phosphate

(GlcN6P). When glucosamine is present at physiologic concentrations, this small molecule binds to the glmS ribozyme and uncovers a latent self-cleavage activity that ultimately leads to degradation of the mRNA [98]. Studies have shown that this system can be utilized within Plasmodium to modulate target gene expression in transgenic 37 parasites as well as used in reverse genetics to explore the function of essential parasite genes without the requirement for promoter modification or protein fusion to the DD.

Levels of the knockdown achieved are comparable to the previously described DD inducible system, and it is believed that when paired together one could achieve an even greater control of the target protein [99].

One approach to understanding fundamental biology and programming new cellar processes in synthetic biology is through synthetic posttranscriptional regulation of gene expression [100]. Previous strategies mentioned above focused on disruption of specific steps within transcription. Strategies that extend the dynamic range of translational and posttranslational control systems are being utilized to increase the flexibility similar to that seen in transcription based regulatory systems which provide the ability to regulate genes at a titrated level appropriate for the specific application

[100]. Synthetic RNA-protein models can be effectively integrated with native cellular mechanisms for controlling translation, and by doing so; more robust regulation of target gene expression can be achieved [101-103]. An existing strategy for achieving synthetic control of eukaryotic translation relies on aptamer-small molecule interactions. Aptamers recognize a specific molecule with high specificity and affinity

[104, 105] and when placed into the non-coding region of an mRNA can mask regulatory sites in the mRNA or become an obstacle for ribosome binding or movement [106]. The aptamer system utilizes the Tet repressor, fused to the development of zygote inhibited protein (DOZI) (TetR-DOZI). In the absence of ATc, the function of DOZI acts to sequester mRNAs which may lead to degradation and translation does not occur. In the 38 presence of ATc, the TetR-DOZI fusion does not bind to the mRNA, permitting translation to occur [107]. The defined aptamer-TetR protein module regulated by tetracycline analogs has shown a substantial reduction in leaky/basal expression while allowing for a more robust regulation of target gene expression to be achieved. Ganesan et al. demonstrated successful integration of this system into Plasmodium, as use of this system established that loss of PfATP4 function is deleterious to blood-stage parasite growth [100].

Proximity Dependent Labeling

Uncovering the function of proteins is often limited to only being fully understood when in context of networks of interactions. Understanding such networks leads to the elucidation of disease processes as well as uncovering new roles for proteins within previously described pathways [108]. While there are techniques in place, such as co-immunoprecipitation or pull down methods and yeast-two hybrid strategies, to search for new molecular associations and protein-protein interactions, these methods display fundamental limitations. Assessing protein interactions in cellular environments different to where they normally occur can lead to incomplete or erroneous datasets due to the lack of proper machinery for posttranslational modifications as well as the normal complement of associated proteins [109].

The promiscuous prokaryotic biotin protein ligase mutant (BirA*) is defective in self-association and DNA binding and displays an affinity for bioAMP two hundred times less than the wild-type enzyme [110, 111]. It has been successfully utilized for spatial labeling and protein isolation in many organisms including the apicomplexan parasite 39

Toxoplasma gondii [109, 112, 113]. To utilize this system, BirA* is fused to a protein of interest. Upon the addition of excess biotin to tissue culture medium, a massive stimulation of promiscuous biotinylation by BirA* non-discriminately biotinylates proteins in its immediate vicinity permitting one-step high-affinity avidin/streptavidin- mediated purification of the tagged protein [114, 115]. This method allows for the detection of potential interactions in their normal cellular environment as well as allowing for the detection of both weak and transient interactions.

Thesis Aims

It has been shown that myosin motors play critical roles within the apicomplexan life cycle. Plasmodium encodes six myosin heavy chains for which only two have a designated light chain partner. The MyoA-MTIP motor is the only molecular motor well characterized in Plasmodium and plays an essential role in invasion, gliding motility and trafficking. Our protein of interest PFF1320c is homologous to the Toxoplasma MLC4 and is annotated in the genome as performing the role of a MLC however, there is currently no evidence to support that this is the actual function of the protein. PFF1320c shows some conserved sequence homology with Plasmodium light chains MTIP, and

MITIPB prompting further exploration into the characterization of this protein through the following aims:

1. Characterize the biological function and phenotypically elucidate the role of

PFF1320c in Plasmodium parasites using alternate knockdown strategies such as

the glmS ribozyme or through the use of a small molecule aptamer. 40

2. Identify and confirm the cognate MHC binding partner through co-

immunoprecipitation studies or through use proximity dependently labeling

using the promiscuous biotin ligase BirA*.

3. Identify and confirm binding of cargo and accessory proteins of the myosin

complex.

Understanding the role of PFF1320c in Plasmodium will provide insight into the growth and development of the parasite as well as provide possible drug targets for future studies.

CHAPTER 2

MATERIALS AND METHODS

Plasmodium Cell Culture And Transfection

All transfections were performed in P. falciparum 3D7attB, NF54attB, or D10attB parasites [116]. Parasites were maintained in RPMI 1640 supplemented with 0.5%

Albumax, 15 mM HEPES, 2 g/L sodium bicarbonate, 50 mg/ml gentamycin, and 1 mg/ml hypoxanthine, and maintained at 5% hematocrit. Parasitemia was determined by

Giemsa smears. Cultures were synchronized with 2 volumes of 0.3 M alanine buffered with 10 mM HEPES (pH 7.5). For experiments requiring tight synchronization, alanine treatment was performed twice at 8-12 h intervals. Transfections were performed on ring stage parasites at 5% parasitemia. After washing 3 times with cytomix, parasites were suspended in cytomix to a 50% hematocrit, mixed with 50-100 µg of plasmid DNA, and electroporated using a Biorad gene pulser (0.31 kV, 960 μFD). All transfections using the 3D7 attB parasites and the pLN vector were also co-transfected with an integrase vector and selected with blasticidin (InvivoGen) and G418 (Cellgro) starting at 48 hours post-transfection. Integration of the transfected plasmid at the GLP3 site was confirmed by PCR.

Western blot

Approximately 2 X105 infected erythrocytes were collected and lysed in 0.5% saponin.

The pellet was resuspended in SDS buffer containing 2% β-mercaptoethanol, and separated by SDS-PAGE. After transfer to a nitrocellulose membrane, blots were blocked in 5% milk, and probed with either mouse anti-GFP-HRP or mouse anti-hemagglutinin-

HRP (HA) epitope (Santa Cruz Biotechnology; 1:20,000 dilution), and developed on film with Pico, Dura, and Super Signal West Femto substrate (ThermoFisher Scientific). 43

Plasmodium aldolase was detected using a rabbit anti-aldolase (Abcam) directly conjugated to HRP (1:40,000 dilution).

Microscopy

Parasites were fixed with 4% formaldehyde and 0.0075% glutaraldehyde overnight.

Parasites were permeabilized with 0.1% Triton-X 100, reduced with 0.1M Tris glycine, and blocked with 5% fetal bovine serum (FBS). The permeabilized parasites were incubated with mouse anti-HA antibody, rabbit anti-HA, or mouse anti-GFP (Santa Cruz) diluted 1:100 in 5% FBS for at least one hour at room temperature and subsequently for one hour with goat anti-mouse IgG-Alexa Fluor 488, donkey anti-rabbit IgG, or Goat anti rabbit IgG TRITC (Jackson Immuno) diluted 1:250. The cells were washed briefly with a

DAPI (4',6-Diamidino-2-phenylindole) solution, mounted in antifade (Invitrogen), and visualized on an Olympus BX60 microscope. Images were analyzed using Slidebook software.

Generation of parasite expression vectors

A full length cDNA encoding PFF1320c and containing a 5’-AvrII site and a 3’-BsiWI site immediately upstream of the STOP codon was amplified from P. falciparum blood stage cDNA using primers 1320-5AV and 1320-3BS (see Appendix I). The resulting 435 bp

AvrII-BsiWI fragment was used to replace the PfENR gene in the plasmid pLN-ENR-GFP (a gift from Dr. David Fidock) to create a PFF1320c-GFP fusion expressed from the constitutive P. falciparum calmodulin promotor. Additionally, a carboxy-terminal triple

HA epitope as a BsiWI-AflII fragment was used to replace the GFP tag to create a 44

PFF1320c-3HA fusion protein. Finally, a P. falciparum coded-optimized E. coli BirA-

R118G (BirA*) gene containing 2 carboxy-terminal HA epitopes with a 5’-BsiWi site and

3’-AflII site was synthesized commercially (Genewiz) and used to replace the GFP tag to create a PFF1320c-BirA* fusion.

To drive expression of these fusion proteins from the native PFF1320c promoter, a 1442 bp fragment corresponding to -1452 to -10 (relative to the ATG start codon) and containing a 5’-ApaI site and a 3’-AvrII site was amplified from P. falciparum 3d7 genomic DNA using primers 1320-5AP and 1320-3AV (see Appendix I). The resulting

ApaI-AvrII fragment containing the PFF1320c promoter was used to replace the Pf calmodulin promoter in the pLN vectors described above (1320-GFP, 1320-3HA and

1320-BirA* fusions).

In an attempt to place expression of PFF1320c-3HA under control of the GlmS ribozyme [99], a 196 bp fragment containing a 5’-SaII site and a 3’-SphI site was commercially synthesized (Genewiz). The resulting SalI-SphI fragment was cloned into the unique SalI and SphI sites in the pLN-PFF1320c-3HA vectors (both calmodulin and native promoters) placing the GlmS ribozyme in the 3’-UTR of the respective gene, 64 bp

3’ of the STOP codon of the HA epitope.

To utilize the TETR-DOZI aptamer vector pMG75, obtained from Drs. Suresh

Ganesan and Joachin Niles, the PFF1320c-3HA or BirA* DNA fragments containing the native promoter were amplified with the following primers, 1320-5AF and 3HA-3AP or

BIRA*-3AP to create DNA fragments with a 5’-AfiII site and a 3’ApaI site. The resulting 45

DNA fragments were used to replace the PfATP4 fragment in the vector pMG75-ATP4

[100].

To delete the genomic copy of the PFF1320oc gene, DNA fragments flanking the coding sequence of the gene were amplified from P. falciparum 3d7 genomic DNA. An

1334 bp upstream fragment, -1566 to -232 relative to the ATG codon, was amplified using primers 1320g-5KA and 1320g-3NC (see Appendix I) and an 1230 bp downstream fragment, +174 to +1404 relative to the STOP codon, was amplified using primers

1320g-SF and 1320g-3SA (see Appendix I). The resulting KasI-NcoI and AfiII-SacII fragments were cloned into the vector pUF-1 [117] and used to transfect P. falciparum parasites as described below. Confirmation of the deletion of the PFF1320c gene was done by PCR analysis using the primers depicted in (Figure 3.1) and listed in Appendix I.

A 2097 bp fragment encompassing the 3’ one-third of the PfMyoC gene as amplified from P. falciparum 3d7 genomic DNA using primers MYOC-5AF and MYOC-3BS

(see Appendix I). The resulting AflII-BsiWI fragment was cloned into the vector pUF-1-

3HA to create a PfMyoC-3HA fusion to transfect and integrate into the genome via a single recombination event. Similarly, a 2011 bp fragment of PfMyoE was amplified from genomic DNA using primers MYOE-5XB and MYOE-3BS (see Appendix I). The resulting XbaI-BsiWI fragment was cloned in the pUF-1-3HA vector using the unique SpeI and BsiWI sites present in that vector.

Full length cDNA clones of PfMyoB and PfMTIP-B containing 5’-AvrII and 3’-BsiWI sites were present in the laboratory (K. Chatterjee and L. Bergman, unpublished results).

The AvrII-BsiWI fragments were used to replace PFF1320c to construct fusions to BirA* 46 in the pLN vector. Subsequently, an 1628 bp fragment encompassing the promoter region of PfMyoB and 1764 bp fragment encompassing the promoter region of PfMTIP-B were amplified from P. falciparum genomic DNA using primers containing a 5’-ApaI site or a 3’-AvrII site. The resulting ApaI-AvrII fragments were used to replace the Pf calmodulin promoter fragment in the pLN-PfMyoB- or PfMTIP-B-BirA* fusion plasmids.

Genomic DNA Isolation From Parasite Pellet

Genomic DNA was isolated from saponin lysted parasite pellets following the DNA purification from blood protocol (QIAamp DNA mini and blood mini handbook). 20 ul of

QIAGEN proteinase K was pipetted into the bottom of a 1.5 ml microcentrifuge tube.

200 ul of the pellet was added on top followed by 200 ul of Buffer AL. The mix was pulse vortexed for 15 seconds and incubated at 56° for 10 minutes. After incubation, 200 ul of

100% ethanol was added to the sample and vortexed again for 15 seconds. The mixture was carefully applied to the QIAamp mini spin column (in a 2ml collection tube) and centrifuged at 8000 rpm for 1 minute. The filtrate was discarded and 500 ul buffer AW1 was added and the tube centrifuged at 8000 rpm for 1 minute. The filtrate was again discarded and 500 ul Buffer AW2 was added and centrifuged at 14000 rpm for 3 minutes. The filtrate was discarded and the column placed in a new collection tube and centrifuged at 14000 rpm for 1 minute to get rid of residual buffer. The column was 47 then placed in a fresh 1.5 ml microcentrifuge tube and DNA eluted in 200 ul of distilled water by centrifugation at 8000 rpm for 1 minute.

Growth Assay for Flow Cytometry

Ring-stage parasites of transgenic and wild type lines were synchronized and diluted to

1% parasitemia and 25 µl of packed infected red blood cells were collected each day for

5 days then analyzed by flow cytometry using the Accuri C6 flow cytometer (BD

Biosciences, San Jose, CA, USA). Cells were stained for nucleic acid content for 30 minutes with 1 mM SYBR Green (Thermo Scientific, Rockfield, IL, USA). Samples were monitored in the FL1 channel for SYBR Green signals. Parasites were selectively analyzed by gating on events with high FL1 signal intensity above or near 105 fluorescent units.

Auto fluorescence of the RBCs was visualized at a lower fluorescent intensity.

Isolation of Biotinylated Proteins

Large cultures (2.5 ml packed red blood cells, 8% parasitemia) were treated for 6 hrs with 150 μM biotin [113], lysed in 0.05% saponin in PBS and stored at -80C. These pellets were solubilized in RIPA Buffer (50 mM Tris-HC1, pH 7.5, 150 mM NaCl, 1% NP-40

0.5% sodium deoxycholate (DOC), 0.1 % SDS) or solubilization buffer (0.5% DOC 1%

Triton X-100, 20 mM Tris pH 8.0, 50 mM NaCl) containing protease inhibitors. After rotating for an hour at room temperature, pellets were incubated on ice for 20 minutes prior to two rounds of sonication (30 seconds with a 30% on, 70% off duty cycle) on a

Fisher Scientific Sonic Dismembrator (Model 500) using a 1/8” microtip. Insoluble 48 material was removed by centrifugation at 14,000 x g. The solubilized supernatant was incubated with avidin-coated magnetic beads (Invitrogen Dynabeads MyOne

Streptavidin C1) overnight at 4°C. The beads were washed 2X with wash buffer 1 (2%

SDS in deionized water), 3X with wash buffer 2A (8.0 M urea, 50 mM Tris pH 7.4, 150 mM NaCl), 2X with wash buffer 2B (50 mM HEPES, pH 7.5, 500 mM NaCL, 1M ethylene diamine tetra acetic acid (EDTA), 1% Triton X-100, 0.1% DOC), 2X with wash buffer 3 (10 mM Tris-HCl pH 8.0, 250 mM Lithium chloride, 1 mM EDTA, 0.5% NP-40, 0.5% DOC)

[109, 111] and 4X with Wash Buffer 4 (50 mM ammonium bicarbonate in deionized water). Each wash constituted a 10 min incubation with tube rotation. Magnetic beads were collected by a 4 min incubation in the magnetic stand. The samples were frozen at

-80o prior to mass spectrometry analysis.

ATc Treatment

Parasites cultures were grown in the presence of RPMI containing 0.5 mM ATc (Sigma) for 24 hours prior to tight synchronization. Cultures were grown to the mid-trophozoite stages (24-30 hrs) and washed 3X with non-ATc containing RPMI. Parasites were then resuspended in non- ATc RPMI and incubated for two hours. 150 μM biotin was added to cultures for 4 hrs, then lysed in 0.05 Saponin in PBS and stored in -80°.

Mass Spectrometry

Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed at the Wistar Proteomics Facility (Philadelphia, PA) under the supervision of Drs. David

Speicher and Hsin-Yao Tang. Samples were digested with trypsin and analyzed using a Q 49

Exactive (QE) Plus or HF mass spectrometer (Thermo Scientific) coupled with a Nano-

ACQUITY UPLC system (Waters). Samples were injected onto a UPLC Symmetry trap column (180 μm i.d. x 2 cm packed with 5 μm C18 resin; Waters), and peptides were separated by RP-HPLC on a BEH C18 nanocapillary analytical column (75 μm i.d. x 25 cm,

1.7 μm particle size; Waters) using a gradient formed by solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). Peptides were eluted at 200 nL/min for 5-30% B over 75 min, 30-80% B over 5 min, constant 80% B for 10 min before returning to 5% B over 1 min. A 30-min blank gradient was run between sample injections to minimize carryover. Eluted peptides were analyzed by the mass spectrometer set to repetitively scan m/z from 400 to 2000. The full MS scan was collected at 70,000 (QE Plus) or 60,000 (QE HF) resolution followed by data-dependent

MS/MS scans at 17,500 (QE Plus) or 15,000 (QE HF) resolution on the 20 most abundant ions exceeding a minimum threshold of 20,000. Peptide match was set as preferred, exclude isotopes option and charge-state screening were enabled to reject singly and unassigned charged ions. MS data were analyzed with MaxQuant 1.5.2.8 software [118].

MS/MS data were searched against the P. falciparum 3D7 PlasmoDB v24 database using full trypsin (K, R) specificity or partial chymotryptic (F, W, Y, L) specificity with up to three missed cleavages, static carboxamidomethylation of Cys, and variable oxidation of

Met, biotinylation of Lys and protein N-terminal acetylation. First search peptide tolerance was set at 20 ppm, main search peptide tolerance at 4.5 ppm, and MS/MS match tolerance at 20 ppm. False discovery was estimated from a reversed sequence decoy database. Consensus identification lists were generated with false discovery rates 50 of 1% at protein and peptide levels. Protein identifications were filtered to remove reverse database entries, common contaminants, and proteins identified by a single razor+unique peptide.

CHAPTER 3

RESULTS

Potential Essential Nature of PFF1320c

Phylogenetic analysis conducted previously demonstrated the Plasmodium MLC,

PFF1320c, is orthologous to Toxoplasma MLC4, however, there is currently no evidence to suggest that either function as a MLC [71]. The lack of the gene encoding PFF1320c in rodent Plasmodium leads to speculation that the gene is not essential for blood stage growth of the parasite. We attempted to knock out (KO) the gene using the pUF-1 vector designed for gene disruption [117]. No parasites were obtained after selection with the positive selection drug DSM-1 for approximately 6 weeks, indicating the possibility of an essential role for this gene within the parasite.

Generation Of Epitope Tagged Transgenic PFF1320c Parasites And Expression In

The Blood Stages.

As there are no working antibodies against endogenous PFF1320c to facilitate fluorescent and immuno-detection, the PFF1320c cDNA was isolated by PCR amplification and the resulting sequence was cloned into a pLN vector containing a 3HA tag from its native promoter as well as a strong constitutive calmodulin promoter. The plasmid was then used to transfect 3D7attB parasites, resulting in integration in chromosome six of the parasite through utilization of the mycobacteriophage recombination system [116] (Figure 3.1). Upon confirming integration at the attB site, western blotting showed strong expression of 1320-HA at its predicted size of ~20kDa in both the over expressed and native transgenic lines using mouse monoloncal anti-HA-

HRP (Figure 3.2).

Figure 3.1: Generation of transgenic PFF1320c using the pLN system. Schematic to represent the pLN based recombination system for integrating tagged genes as a second copy in the GLP3 locus of P. falciparum genome

Figure 3.2: Successful expression of tagged PFF1320c in blood stage parasites. Western Blot showing 1320-HA expression from the Native (A) and Calmodulin (B) promoters.

Figure 3.3: PFF1320c is essential for blood stage growth. A. The pUF-1 derived knock out plasmid used to disrupt the endogenous PFF1320c gene and the predicted PFF1320c locus after double cross over integration is shown. Small arrows indicate the binding sites of primers used in PCR reactions to confirm the knock out with numbers. B. PCR confirming knockout of endogenous PFF1320c. Primers 1.) 1320-233F 2.) pUF1-2068R 3.) 1320-2686R 4.) 1320-2910F 5.) pUF1-4035F 6.) 1320-4286R

Using these transgenic parasites we were only able to achieve successful KO of the endogenous copy of PFF1320c when a HA-tagged copy was expressed from an exogenous chromosome six site, thus ruling out the possibility of the locus being inaccessible for deletion. This also demonstrates that addition of the 3HA tag to the C- terminus of PFF1320c is functional as it complements the deletion of the genomic copy of the gene (Figure 3.3).

The expression pattern of PFF1320c in different stages was analyzed in the

3D7attB stain of P. falciparum using 1320-HA parasites under the control of the native promoter. The parasites were tightly synchronized and samples collected every 8 hrs post invasion (pi) to analyze the change in protein expression. 1320-HA is expressed throughout the blood stages of Plasmodium with expression first being observed in the very early ring stages (3-10 hrs pi) with maximum expression observed during the mid to late trophozoite stage (27-34 hrs pi) (Figure 3.4A). This was in agreement with its mRNA expression profile on PlasmoDB (Figure 3.4B) [119]. These data lead us to conclude that due to the difference in expression profile, at both the transcriptional and translational levels, this suggest that PFF1320 does not bind to the MyoA or MyoB MHC and does not play a role in invasion of the parasite into the red blood.

Cellular Distribution of PFF1320c

Immunofluorescence assays (IFA) were conducted to determine the location of

PFF1320c using an anti- HA antibody to stain PFF1320c and DAPI to stain the nucleus.

No specific fluorescence was detected using the anti-HA antibody with methanol/acetone fixation so IFA’s were done using glutaraldehyde/formaldehyde 58 fixation. In contrast to MTIP and MTIP-B, which are found localized to the periphery of merozoites and apex of rhoptries respectively, 1320-HA is uniformly distributed within cytoplasm of the Plasmodium parasite with punctate areas seen throughout (Figure

3.5).

Co-Localization of PFF1320c and PfMyoC

As we believe PFF1320c to be the MLC component of a myosin motor, we attempted to generate transgenic strains of Plasmodium MHCs expressing either an HA or GFP C- terminal tag, which we would then use for co-immunoprecipitation and/or co immunofluorescence assays. Of the four MHCs, PfMyoC, PfMyoD, PfMyoE, and PfMyoF we were only able to successfully generate PfMyoC and PfMyoE transgenic parasites expressing an HA tag, however we’ve ruled out PfMyoE as a binding partner for

PFF1320c due to its expression being found towards the very end of the lifecycle in the blood stages. We then co-transfected PfMyoC-HA with a 1320-GFP to create a transgenic parasite strain that expresses a tagged version of both PFF1320c and

PfMyoC. Using tightly synchronized parasites we collected samples during times points when both proteins are maximally expressed and performed co-immunoprecipitation assays. Though we were successfully able to detect 1320-GFP via western analysis we were unfortunately unable to detect PfMyoC-HA suggesting that PfMyoC maybe insoluble indicative of the association of this motor with a detergent resistant fraction of the parasite (data not shown). Subsequently, we were able to perform co- immunofluorescence using PfMyoC-HA/1320-GFP parasites though results were not 59 conclusive due to the inconsistent pattern of overlap observed in samples collected at time points when expected co-expression should occur (Figure 3.6).

Knockdown Of PFF1320c Does Not Show an Effect on Growth of the Parasite.

Initial attempts at observing the phenotype of PFF1320c by knocking out the endogenous locus alone via traditional methods have failed. Attempts at knocking down

PFF1320c within the parasite using the FKBP and DHFR inducible protein degradation domain have also been unsuccessful due to our inability to achieve integration into the attB site and failure to see complete degradation with other protein-DDs (Jenkins and

Bergman, personal communication). We next explored an alternative knockdown system using the glmS ribozyme. Through utilization of this system, we can control the expression of our P. falciparum gene of interest by the target gene bearing a glmS ribozyme in the 3’ translated region (UTR). Upon addition of the glucosamine substrate the glmS ribozyme becomes activated, thus cleaving the 3’ end of the mRNA resulting in degradation of the remaining mRNA by other nucleases, which allow for efficient knockdown in transgenic P. falciparum parasites (Figure 3.7).

Using the pLN system, we generated a parasite strain overexpressing 1320-HA- glmS with the calmodulin promoter from the attB site and obtained parasites by limiting dilution. The tagged PFF1320c was expressed at ~20Kd as seen by western blot (Figure

3.8A) To obtain knockdown, 1320-HA-glmS parasites were treated with 1.25 mM of glucosamine or left untreated for 24 hrs and then saponin lysed for western analysis.

The parasites treated with glucosamine showed successful knockdown of protein expression as compared to the control (Figure 3.8B). 60

Figure 3.4: PFF1320c is expressed during the trophozoite stage in the blood stage of the parasite. A. Western blot of tightly synchronized parasites showing stage specific expression of PFF1320c. B. PlasmoDB mRNA expression profile of PFF1320c [119].

Figure 3.5: PFF1320c is localized to the cytosol of the parasite. 1320-HA expressed from the native promoter localized to the cytosol. Nuclear staining using DAPI in blue.

Figure 3.6 Co-immunofluorescence of PFF1320c and PfMyoC. IFA of transgenic parasites expressing 1320-GFP and MyoC-HA. Nuclear staining using DAPI blue.

Upon observation of successful knockdown through utilization of the glmS ribozyme, we then knocked out the endogenous copy of the PFF1320c gene on chromosome six using the pUF-1-based strategy described above resulting in the control of the PFF1320c by the glmS ribozyme (Figure 3.9A&B). Knockout parasites overexpressing 1320-HAglmS were grown in media in the absence of glucosamine or containing varying levels of glucosamine for 72 hrs. Western analysis shows that knockdown of protein expression was achieved in parasites incubated with 2.5 mM and 5 mM of glucosamine (Figure

3.9C). We then monitored growth of the 1320-HA-glmS-KO parasites with and without glucosamine for several invasion cycles and compared them to the 3D7attB wild type control. The parasites grew at the same rate with and without the glucosamine knockdown (Figure 3.10). This indicates that the addition of glucosamine does not cause a defect in growth to either the wild type line or transgenic line and that knockdown of

1320-HAglmS-KO in the 3D7attB line was not detrimental to the parasite. This may be due to the level of PFF1320c remaining after glucosamine knockdown being sufficient to support the parasite. In an initial attempt, we were unable to obtain 1320-glmS-HA-KO parasites expressed from the native promoter.

The TetR-DOZI Aptamer System Enhances Regulation of PFF1320C.

To continue our efforts to investigate the role of PFF1320c within the blood stages of the parasite, we utilized an existing strategy that relies on aptamer-small molecule interactions for achieving synthetic control of eukaryotic translation. In the presence of DOZI, proteins and certain mRNAs are converted into quiescent messenger ribonucleoprotein particles where transcripts are stored for translation at a later time 64

[107]. The aptamer system utilizes the Tet repressor, fused to the DOZI (TetR-DOZI).

Disruption of DOZI deregulates the stability of many transcripts normally repressed during the asexual stage of development in blood. In the aptamer system, the small molecule ATc regulates the interaction between the Tet repressor and the aptamer, which is found in the untranslated 3’ region of the gene. In the presence of ATc, the mRNA is translated and in the absence of ATc is turned off [100]. Using the pLN system we generated a transgenic parasite strain expressing 1320-HA-Aptamer from its native promoter in D10attB parasites (Figure 3.11). The tagged 1320-HA-Aptamer was expressed at ~20KDa. We first assessed whether the aptamer system functions to regulate translation of 1320-HA-Apatmer by growing parasites along with wild type parasites in the absence or presence of ATc for 48 hrs. Using western blot analysis, we showed successful ATc inducible regulation of gene expression in the 1320-HA-Aptamer parasites (Figure 3.12).

To prove that the PFF1320c gene is indeed regulated in response to ATc, we synchronized parasites and put them in the presence or absence of media containing

ATc during the ring stages of the life cycle and allowed the parasites to grow until the beginning of the trophozoite stage. Parasites were then split into new flask and put in the presence or absence of ATc for the remainder of the trophozoite stage (12 hrs) then collected for western analysis. Parasites that were initially treated with non-ATc containing media and then put in the presence of ATc upon being split showed ATc regulated expression of the 1320-HA-Aptamer gene as compared to loss of expression seen in parasites that were initially in the presence of ATc and then placed into non-ATc 65

Figure 3.7: Schematic of the glmS ribozyme reverse genetic tool. The ribozyme is inserted in the 3’-UTR after the coding region so that it’s present in the expressed mRNA. The inducer, glucosamine, binds to the ribozyme causing the mRNA to self cleave resulting in degradation of the mRNA and knockdown of protein expression [99].

Figure 3.8: PFF1320c is knocked down in the presence of Glucosamine. A. Western blot showing expression of 1320-HA-glms from. B. Knockdown (KD) of 1320-HA-glmS expression in transgenic parasites incubated with glucosamine for 24hrs

Figure 3.9: Succesful knockdown of PFF1320 in the 1320-HA-glmS KO transgenic parasite line upon addition of glucosamine substrate. A. Western blot showing expression of 1320-HA-glmS-KO from the calmodulin promoter. B. PCR confirming knockout of endogenous PFF1320c. See Primers on Figure 3.3B (A) 1+2 (B) 4+6 (C) 5+6 C. Western blot showing 1320-HA-glmS-KO knockdown in the presence of up to 5 mM glucosamine for 72 hrs.

Conditonal Growth in 5 mM Glucosamine

12 3D7aB - 10 3D7aB + 8 1320-glmS-KO - % Parasitemia 6 1320-glmS-KO + 4

0 Day 1 Day 2 Day 3 Day 4 Day 5

Figure 3.10: Growth of transgenic parasites are not effected by glucosamine mediated knockdown. Wildtype (3D7attB) and the transgenic (1320-HA-glmS-KO) parasites were incubated with or without 5 mM glucosamine for 5 days. Samples were collected by formaldehyde fixation at 4°C and stained with SYBR green for a single channel flow cytometry.

69 containing media upon split (Figure 3.13). This data confirms that through the aptamer system, we can stringently control the expression of PFF1320c. To more tightly regulate and control the expression of PFF1320c, we further plan to knockout the endogenous copy of the PFF1320c gene using the CRISPR-Cas9 system.

Biotinylation In 1320-BirA* Parasites Is Distinct from Cytoplasmic BirA* and

Localizes to the Cytoplasm.

The MyoA MTIP motor complex is the only myosin motor that has been very well characterized within Plasmodium. Using the neck and tail domains of MTIP as bait it was confirmed to be the light chain partner of PyMyoA by a yeast two-hybrid screen using a

P. yoelii cDNA library for screening [46]. MTIP is also associated with the glideosome complex GAP45, which has shown to be essential for gliding motility and recruits the

MyoA-MTIP motor to the IMC. Attempts to identify the MHC binding partner of

PFF1320c using the Yeast Two Hybrid (Y2H) system were unsuccessful due to the auto activation of reporter genes by the DNA binding domain PFF1320c fusion protein alone

(L. Bergman, personal communication). In an attempt to determine the MHC binding partner along with other proteins that could comprise the myosin motor complex, we utilized the promiscuous prokaryotic ligase BirA*, which non-discriminately biotinylates proteins in its immediate vicinity. Using the pLN system we tagged the carboxy– terminus of PFF1320c with BirA*, codon optimized for Plasmodium expression, as well as 2HA epitopes for protein detection from its native and from a robust calmodulin promoter. We also generated parasites expressing an unconjugated BirA*.

Figure 3.11: Conditional knockdown with TetR-Dozi apatmer. pMG75 plasmid was used to transfect the P.falciparum strain D10attB resulting in integration of the tagged allele on chromosome 6 of the parasite. A similar plasmid containing PFF1320c fusion to BirA* (with the aptamer) has been utilized.

Figure 3.12: The aptamer system regulates translation of 1320-HA-Aptamer transgenic parasites. 1320-HA-Aptamer transgenic parasites grown in the presence of ATc for 48 hrs show induced expression.

Figure 3.13 Confirmation of aptamer induced regulation of translation in 1320-HA- Aptamer transgenic parasites. A. Experimental outline of aptamer induced regulation. B. Western blot confirming 1320-HA-aptamer induced regulation of translation.

PRO-1320-BirA* was expressed at the expected size ~45 KDa and the unconjugated

BirA*-HA at 28 KDa as seen by western analysis (Figure 3.14A). IFA confirmed localization of 1320-BirA* expressed from both the native and calmodulin promoter to the cytoplasm of the parasite (Figure 3.14B).

To determine whether the BirA* tag was functional within the parasites we treated parasite lines with 150 μM biotin. Synchronized parasites were incubated in biotin for 4-12 hrs during the trophozoite stage to analyze the most efficient time for maximum biotinylation. Western blotting of saponin-lysed pellets with streptavidin-HRP conjugate revealed significant biotinylation in each time point. Most importantly, PRO-

PFF1320-BirA* exhibited a unique pattern of biotinylation as compared to unfused BirA*

(Figure 3.15). The decreased overall level of biotinylation observed in the PRO-PFF1320-

BirA* parasites is consistent with the lower level of expression of the fusion protein seen with its native promoter. Through IFA, we confirmed that biotinylation was cytoplasmic (Figure 3.16). Together, these data indicate that 1320-BirA* causes specific and consistent biotinylation within the cytoplasm. We then performed biotinylation assays where parasites were tightly synchronized and then incubated with 150 μM biotin starting at 24 hrs post invasion. Samples were collected at 32 hrs post invasion for western analysis and mass spectrometry (Figure 3.17).

Mass spectrometry was performed on independent samples of PFF1320-BirA* that will be discussed later, as we have developed a system to regulate the expression of the 1320-BirA fusion protein. 74

Using the aptamer system described above, we generated transgenic parasites expressing 1320-BirA*-Aptamer from its native promoter. To ensure the functionality of the aptamer, parasites were placed in ATc containing media or non-ATc containing media for 48 hrs. Samples were collected and observed for knockdown via western analysis. Parasites in the presence of ATc showed an increased expression of the 1320-

BirA* fusion (Figure 3.18A). This is confirmed by IFA where parasites grown in the presence of ATc for 24 hrs showed an increased expression of 1320-BirA*-Aptamer as compared to those parasites in non-ATc media leading to the conclusion that with the small molecule ATc, we can stringently control the expression of 1320-BirA*-Aptamer

(Figure 3.18B). To build on this observation, we then tightly synchronized 1320-BirA*-

Apatamer and wild type 3D7attB parasites and then incubated the parasites in media containing ATc. At 24 hrs post invasion, parasites were washed with non-ATc containing media and then incubated for an additional 2 hrs in non-ATc media. 150 μM biotin was then added to the cultures and allowed to incubate for 4 hrs. Samples were then collected and mass spectrometry performed on two independent samples (Figure 3.19).

The complete lists of proteins identified can be found in the appendix. To filter out the background from non-specific biotin labeling or binding to the beads, we analyzed the datasets for significance based on proteins identified by three or more peptides that represented at least 5% sequence coverage of the protein as summarized in (Table 3.1).

When we examined our collective data sets for common proteins we found substantial

(but not complete overlap) between the two data sets (Figure 3.20). Importantly, in each case, there were substantially more proteins found to be significantly enriched in 75 the samples isolated from the parasites expressing the PFF1320c-BirA* fusion protein.

Although paired samples are matched for approximate equal parasitemia prior to lysis and for approximate equal protein concentrations in the lysates, we were uncertain as to why the total number of proteins identified in the two independent runs differs. As we presume PFF1320c to be a MLC, were surprised to find that an exclusion of MHCs among the similar proteins. Though we pulled down PfMyoC, it did not meet our set parameters for specific inclusion, though large in size, possibly due to the nature of its solubility (Table 3.2). Between the two experiments 14 proteins were identified in both replicates (Figure 3.21) including PFF1320c, which is expected as a BirA* fusion would biotinylate itself. (Table 3.3). Among the proteins that interact with 1320-BirA*-Aptamer the top two hits after PFF1320c were subunits of the T-complex family of chaperonin proteins, subunit beta and subunit gamma. We next used the PlasmoDB database to explore the additional properties of the 14 proteins identified in the 1320-BirA*-

Aptamer samples. Five of the 14 proteins, including the two mentioned above, belonged to the T-complex family of chaperonin proteins. An additional 7 proteins, found in each separate run, plus one found in both, had GO Functions and GO Components related to the Food Vacuole, an organelle of interest as the PFF1320c expression profile corresponds accordingly with food vacuole formation and hemoglobin uptake. The significance of these findings will be discussed below.

Figure 3.14: Expression and localization of 1320-BirA*-HA. A. Western blot showing expressing of PRO-1320-BirA*-HA and BirA*-HA. B. IFA localization of 1320-BirA*-HA from the native and Calmodulin promoter. Nuclear staining using DAPI blue.

Figure 3.15: 1320-BirA* exhibits a unique biotinylation profile. Western blot showing detection of biotinylated proteins from saponin lysed pellets of 1320-BirA* parasites expressed from its native (PRO) or calmodulin promoter.

Figure 3.16: 1320-BirA* biotinylation is localized to the cytosol. Fluorescence assay of BirA* expressing parasites treated with biotin for 12 hrs and probed with streptavidin conjugated alexafluor-488. Nuclear staining using DAPI blue.

Figure 3.17: 1320-HA-BirA* Biotinylation. A. Schematic of experimental timeline. B. Western blot of samples collected from parasites incubated with 150 μM biotin and probed with streptavidin-HRP.

The Identification of Presumed Myosin Complex Proteins through Mass

Spectrometry of MyoB-BirA* and MTIPB-BirA* Pulldowns.

Due to the results observed with 1320-BirA* where no MHC was identified, we sought to explore a more characterized myosin motor complex. The MLC PfMTIPB was recently shown to be the binding partner of the MHC PfMyoB and it is bound to the extreme C-terminus of MyoB. Inability to knockout the genes encoding PfMTIPB and

PfMyoB indicates an essential role for these genes within the parasite. There is a significant size variability seen amongst the MLC of Plasmodium with PfMTIPB being the largest at 652 AA, making it the largest MLC to be identified in any species. Both MyoB and MTIPB are expressed >40 hrs pi and are localized to the apex of merozoites and the rhoptries respectively, though the role of this myosin motor complex has yet to be deduced. To further investigate the role of this myosin motor complex as well as discover potential binding partners that are common among both PfMyoB and PFMTIPB, we preformed proximity dependent labeling through use of the promiscuous BirA* ligase. Using the pLN system, we tagged the C–terminus of PfMyoB and PfMTIPB with

BirA* from the native promoters. To perform proximity dependent labeling, both parasite lines were tightly synchronized and treated with 150 μM biotin for 6 hrs during maximal expression as seen in PlasmoDB expression profile. Western blotting of saponin-lysed pellets with streptavidin-HRP conjugate revealed significant difference in the biotinylation profiles of both the MyoB-BirA*-HA and MTIP-BirA*-HA, as compared 81 to the wild type control 3D7attB. Immunofluorescence shows localization of biotinylated proteins to be distributed cytoplasmically though overlay with HA staining for biotinylated proteins shows a presumed colocalization with MyoB and MTIPB (Figure

3.22).

Mass spectrometry has been performed on PfMyoB-BirA*-HA and PfMTIPB-

BirA*-HA parasites treated with 150 uM biotin for six hours, 32-42 hrs pi. Biotin treated

3D7attB parasites were used as a control (Figure 3.23). It should be noted that significantly more biotinylated species were found in the pellet fraction (insoluble after

14,000 x g spin) as compared to previous studies with PFF1320c-BirA*. To filter out the background, the datasets from the MyoB, MTIPB and 3D7attB parasites were analyzed for significance based on proteins identified by three or more peptides that represented at least 5% sequence coverage of the protein. The complete list of proteins can be found in (Appendix III) and a summary in Table 3.4.

Over 38.8% (386) of proteins identified were found to be specific to both

PfMyoB-BirA* and PfMTIPB-BirA* (Figure 3.24) while additional proteins were found to be unique for either the MyoB or MTIP BirA* fusion expression parasites. All proteins observed in the 3D7attB sample were also found in the other two samples. We next used the PlasmoDB database to explore additional properties of the 386 proteins.

Twenty-one proteins of interest were identified based on their functional properties that spanned our area of interest. These areas included proteins related to motor activity, actin binding, calcium binding, and cytoplasmic microtubule organization. We are also interested in proteins involved in DNA replication and nuclear segregation as 82

PfMyoB and PfMTIPB are both expressed during the later stages of the parasites life cycle and could potentially be involved in schizogeny (Appendix IV). The significance of these findings will be discussed below.

Figure 3.18: The small molecule ATc stringently controls the expression of 1320-BirA*- Aptamer. A. Western blot 1320-BirA*-Aptamer transgenic parasites incubated with ATc for 48 hrs. B. IFA of 1320-BirA*-Aptamer transgenic parasites in the presence or absence of ATc for 24 hrs.

Figure 3.19 Biotinylation in 1320-BirA*-Aptamer parasites. A. Schematic of experimental design B. Western blot of 1320-BirA*-HA expressing parasites. C. Western blot showing detection of biotinylated proteins from saponin lysed pellets treated with 150 μM biotin for 4 hrs. The * indicates a non-specific band observed in this particular sample using a new reagent (Super Signal West Dura Chemiluminescent Substrate). This band was not observed previously.

Sample Total Proteins >2 peptides proteins >5% sequence coverage identified 3D7attB Run 1 150 64 3D7attB Run 2 601 358 1320-BirA*-Aptamer Run 1 248 113 1320-BirA*-Aptamer Run 2 663 453

Table 3.1: Proteins identified by mass spectrometry in 1320-BirA* samples. Both total numbers and significant detection are shown.

Figure 3.20: Proteins identified in parasites expressing BirA* fusion proteins. Venn diagrams comparing proteins detected at significant levels between control and BirA* parasites in two separate runs.

PF3D7_1329100 Myo C Peptide Sequence Coverage 1320-BirA*- Aptamer Peptides Run 2 4 3%

3D7attB Peptides Run 2 5 4%

Table 3.2 PfMyoC proteins found in biotinylation. Peptides and sequence coverage of PfMyoC proteins found in biotinylation data sets.

Figure 3.21: Proteins from 1320-BirA*-Aptamer pulldowns identified by mass spectrometry. Venn diagram showing overlay of the proteins found common among the two 1320-BirA-Aptamer samples. Proteins were unique to 1320-BirA-Aptamer samples vs. 3D7attB samples in each case.

Gene ID Product Molecular B. C D E. F. G. H. I. Description Weight PF3D7_0627200 Myosin Light 16.143 10 0 89.9 0 11 0 92.1 0 Chain PF3D7_0306800 T-complex Protein 59.198 10 0 28.5 0 21 1 56.5 3.6 Beta Subunit PF3D7_1229500 T-complex Protein 60.662 7 0 14.8 0 8 2 23.1 6.5 1 Gamma Subunit PF3D7_0904800 replication 56.234 6 0 16.5 0 12 0 36.2 0 protein A1, small fragrament (RPA1) PF3D7_0419600 RAN BINDING 33.195 5 0 20.4 0 5 0 19.6 0 PROTEIN 1, PUTATIVE PF3D7_1412600 DEOXYHYPUSINE 57.241 5 0 13.1 0 18 0 42.5 0 SYNTHASE (DHS) PF3D7_0810800 dihydropteroate 83.373 5 0 8.6 0 4 2 9.2 4.8 synthetase (DHPS) PF3D7_1138500 protein 105.4 5 0 6.2 0 3 0 6.3 0 phosphatase 2C (PP2C) PF3D7_1020900 ADP-ribosylation 20.912 4 2 32.6 13.8 4 1 37.6 9.9 factor (ARF1) PF3D7_1212000 glutathione 23.952 3 0 26.8 0 7 1 41.5 7.8 peroxidase-like thioredoxin peroxidase (TPx(Gl)) 205 PF3D7_1132200 TCP-1/cpn60 60.26 3 0 10.5 0 5 1 16.2 2.8 chaperonin family, putative Alpha PF3D7_1457300 conserved 75.986 3 0 8 0 3 0 5 0 Plasmodium protein, unknown function PF3D7_0608700 T Complex Protein 61.533 3 0 7 0 9 1 21.4 3.9 Zeta PF3D7_0214000 T-complex protein 60.958 3 0 6.1 0 4 0 11.1 0 1, putative Theta

Table 3.3: Proteins found similar in data sets from 1320-BirA*-Aptamer. Proteins identified as being specific to 1320-BirA*-Aptamer found in both pull downs. Number of peptides and sequence coverage shown. A.) Molecular Weight B.) 1320-BirA*-Aptamer peptides run 1. C.) 3D7attB Peptides Run 1 D.) 1320-BirA*-Aptamer sequence coverage Run 1. E.) 3D7attB Sequence Coverage Run 1 F.) 1320-BirA*-Aptamer peptides run 2. G.) 3D7attB Peptides Run 2 H.) 1320- BirA*-Aptamer sequence coverage run 2. I.) 3D7attB sequence coverage Run 2

Figure 3.22: Biotinylated proteins co-localize with MyoB and MTIPB BirA* fusions. IFA of parasites incubated with biotin for 6 hrs show co-localization with biotinylated proteins.

Figure 3.23: Biotinylation of MyoB and MTIPB BirA* fusion. Western blot of samples collected from parasites incubated with 150 μM biotin and probed with streptavidin- HRP.

Sample Total proteins Proteins >2 peptides identified >5% sequence coverage

3D7attB 621 312 MyoB-BirA* 1335 926 MTIPB-BirA* 1240 761

Table 3.4 PfMyoB and PfMTIPB associated proteins identified by mass spectrometry. Both total numbers and significant detection are shown.

Figure 3.24: Proteins from MyoB and MTIPB BirA* fusion pulls downs. Venn diagram of proteins identified in all three BirA* samples.

CHAPTER 4

DISCUSSION/CONCLUSION

MLCs play an integral role in mediating the association of a MHC binding partner or determining the intracellular localization of the myosin motor complex. Toxoplasma boast seven MLCs with redundancy seen among TgMLC3, TgMLC5, and TgMLC7 for binding to TgMyoH while to date Plasmodium only has three presumptive MLCs implying the likelihood for multiple motor systems. Of the three MLC’s identified in

Plasmodium only the role of MTIP, has been greatly characterized as playing an essential role in gliding motility, invasion, and trafficking [46, 52, 79] Despite PFF1320c’s annotation in the genome as performing the role of a MLC, and its orthologous nature to Toxoplasma’s MLC4 [71] there is still very little evidence to support its biochemical role. Unlike MTIP or MTIPB, the PFF1320c gene is not found in rodent infecting plasmodia. Genetic disruption of the gene using traditional and conditional knockdown strategies has been unsuccessful indicating a potential essential role in the asexual blood stages. Sometimes genes can be refractory to being deleted from the genome as their loci may be inaccessible. We have eliminated this doubt by showing that when

PFF1320c is expressed from a different chromosome, we can knock out the endogenous gene encoding PFF1320c. Although this suggests that the gene is truly essential, it has been suggested that the stability of each component within a myosin motor relies on the interaction between the light and heavy chain, indicating that light chains are only stable in the presence of their corresponding heavy chain, which may aid in explaining our inability to delete the gene without knowing the MHC binding partner [71].

Through the work of this dissertation, we set out to characterize the role

PFF1320c, as well as find its cognate MHC binding partner. Using the mycobacteria 99 phage recombination system, we generated a 3HA fusion to the C-terminal end and then deleted the endogenous copy of the gene. Using this strain, we explored its expression and localization, which can contribute greatly to elucidating the function of a gene. PFF1320c is found localized to the cytoplasm of the parasite with punctate staining seen throughout under the control of its native promoter. Interestingly, expression of the gene coincides with the highly dynamic mid to late trophozoite stages of the parasite, which is very similar to its mRNA profile, where several biological processes such as hemoglobin uptake, intake of nutrients from the surrounding areas, as well as the rapid formation of organelles that could utilize a myosin motor complex occur [120]. Consequently, expression of the protein during this stage maybe excludes its involvement in egress or invasion of the parasite as well as eliminates the possibility of PFF1320c being an alternative light chain for PfMyoA or PfMyoB.

Plasmodium degrades 80% of the erythrocyte hemoglobin [121] via four distinct phases that can be unambiguously distinguished from one another by morphology and timing. Two of the pathways, the “big gulp” and phagotrophy, both deliver hemoglobin in bulk from the erythrocyte cytosol to the parasite in an actin independent manner, but they occur at separate ends of the parasite’s life cycle. Vesicular requirements for delivering hemoglobin and synthesized proteolytic enzymes transported to the food vacuole (FV) are much different, and to address the need for continual transport of these enzymes to the FV, small hemoglobin vacuoles (SHV) and cystostomal tubes derived from cytostomes are likely employed. However there is still limited information on mechanisms involved in the trafficking process [122]. Cytostomes are defined as a 100 localized invagination of the parasite outer membrane, the parasitophorous vacuolar membrane (PVM) and the parasite plasma membrane (PPM) with an electron dense collar associated with the neck of the cytostome [123, 124]. The resulting transfer of hemoglobin is an actin-dependent process. SHV’s are the most commonly occurring hemoglobin vacuoles (HV) in trophozoites and schizonts and overall hemoglobin uptake from these SHV’s increases from <8% in ring stage parasites to <50% in trophozoites and schizonts [125]. When treated with actin inhibitors, Elliot et. al, observed an increase in internalized hemoglobin volume and decreased FV volume suggesting inhibition of transport of HVs to the FV [125]. This implies that HVs are transported to the FV by complexes that utilize actin and pressumably contain molecular motors and due to the implied essentiality of PFF1320c, this MLC may play a role in this process. This hypothesis is supported by studies from Milani et.al., that confirm in the presence of

BDM, a myosin ATPase inhibitor, and the protease E64, infected red blood cells showed food vacuoles devoid of hemoglobin suggesting that cytostomes use an actin-myosin motor to transport hemoglobin to the FV [122]. To test PFF1320c’s involvement, we can perform conditional knockdown strategies to observe the change in hemoglobin, food vacuole volume, and cytostome morphology.

Conditional knockdown methods to observe the phenotype of the Plasmodium parasite in the absence of PFF1320 utilizing the FKBP destabilization domain and the

DHFR degradation domain have been unsuccessful. Therefore, we generated a transgenic parasite line that allowed conditional knockdown of the PFF1320 gene through use of a regulated ribozyme. The ribozyme knockdown system involves the 101 glmS bacterial ribozyme sequence inserted into the 3’ UTR of the gene of interest [98].

The glmS ribozyme is inactive without glucosamine and upon its addition, the ribozyme is activated cleaving the 3’UTR in the mRNA transcript in which it resides. This removes the polyA sequence from the mRNA leading to degradation of the remainder of mRNA.

The glmS ribozyme was incorporated into the 3’ UTR of the PFF1320c gene expressed from the attB site, but from a strong constitutive calmodulin promoter. We then deleted the endogenous copy of the gene so that regulation of the gene would be solely through the glmS ribozyme. Using concentrations up to 5 mM for our studies, we saw that the addition of the glucosamine led to a significant knock down of PFF1320c protein at 5 mM and an observed knockdown at 2.5 mM (Figure 3.9). This reduction in protein expression had no detrimental effect on parasite growth as seen when parasites were grown in the presence of 5 mM glucosamine for several cycles (Figure 3.10). This data strongly suggests that only a small amount of PFF1320 is necessary for growth and survival during the blood stages and prevents us from drawing conclusions about possible roles that PFF1320c could play in the parasite. Attempts are being made to integrate the glmS ribozyme at the endogenous PFF1320c locus using a single recombination pUF-1 based vector. As discussed below, preliminary results have suggested that the TETR-DOZI aptamer system provides tight regulated control of expression.

Much remains to be discovered about PFF1320c. We’ve hypothesized that this protein functions as MLC and is bound to a MHC, which makes up a myosin complex of unknown function, and have attempted several methods to identify proximal proteins, 102 or specific interacting proteins. Our first attempts at identifying potential binding partners through use of the Y2H system were unsuccessful due to the auto activating nature of the reporter gene. We next tried immunoprecipitation of PFF1320c with α-HA antibody conjugated to agarose beads. However, results from these pull downs were found to be inefficient, preventing us from identifying potential interacting partners. We then tried generating transgenic strains of Plasmodium MHCs expressing either an HA or

GFP C-terminal tag, which we would then use for co-immunoprecipitation and/or co immunofluorescence assays. We were only able to successfully generate PfMyoC and

PfMyoE transgenic parasites expressing an HA tag, however we’ve ruled out PfMyoE as a binding partner for PFF1320c due to its expression primarily occurring at very end of the lifecycle in the blood stages. However, we can utilize PfMyoE in future experiments that would allow us to deduce its MLC binding partner. Our attempts at co- immunoprecipitation using parasites expressing 1320-GFP and MyoC-HA were unsuccessful. Our inability to detect MyoC-HA may be due to its presumptive insoluble properties indicative of the association of this motor with a detergent resistant fraction of the parasite. To confirm this observation, we can perform subcellular fractionation of

PfMyoC-HA, which may lead to critical information regarding the cellular location of the protein within the parasite. Our co-localization attempts were inconclusive suggesting that the binding of PFF1320c and PfMyoC may not be long lived or may not exactly occur when both proteins are maximally expressed. Thus it may be beneficial to collect samples in smaller intervals to allow for maximum coverage of the times when co- localization may occur. 103

As traditional methods to elucidate potential binding partners have been unsuccessful, we undertook the approach called BioID, a form of proximity dependent labeling, which utilizes the promiscuous biotin ligase BirA*. BirA* non-discriminately biotinylates proteins in its immediate vicinity which in turn allows for the identification of proximal proteins through affinity purification and mass spectrometry. The promiscuous biotin ligase BirA* contains an R118G mutation which catalyzes the formation of biotin-AMP, thus activating the biotin. This point mutation causes a reduced affinity of the enzyme for biotin-AMP, causing it to be released where it can react with lysine residues within an estimated range of 20-30 nanometers [109] This system is advantageous in the fact that not only can it identify direct interactions, but also proteins that are nearby or interact loosely or transiently.

Expression of PFF1320c fused to BirA* from the attb site under the control of both the native and calmodulin promoter showed robust expression and localization to the cytosol. When incubated for 8 hrs with biotin and analyzed the biotinylated bands from both the native and overexpressed 1320-BirA*-HA samples showed a unique biotinylation profile as compared to the control (BirA* alone) when probed with streptavidin-HRP. While this system proved useful in providing information on potential interacting proteins there were several limitations. First, the sheer number of proteins identified made it difficult to draw specific conclusions on the function and potential binding partners of PFF1320c. We believe that continuous expression of 1320-BirA*-HA in the presence of biotin may label proteins throughout its life in the asexual stages that don’t necessarily play a role in the biological function of the protein. It could be 104 assumed that a shorter labeling time would help to circumvent this, however, the amount of biotinylation seen in the 4 hr incubation as compared to the 8 hr incubation when expressed from the native promoter appears to be comparable (Figure 3.15). The next limitation we ran into involved the number of proteins found in the background of the control sample. The number of proteins biotinylated in Plasmodium is very low indicating that there could be a population of proteins binding non-specifically to the beads and not necessarily through biotinylation. We believe that this is likely due to the charge interactions between basic proteins and the acidic nature of the beads and that more washes or longer washes may help to reduce this background.

To counteract this issue, we next explored a system that would allow for a more controlled regulation of protein expression, thus allowing us the ability to limit BirA* labeling in 1320-BirA*-HA parasites. By inducing expression for a specific amount of time, we could capture critical protein interactions while possibly reducing the amount of background detected significantly. Through use of the aptamer system, it has been shown that we can achieve synthetic control of eukaryotic translation in Plasmodium.

The aptamer system utilizes the Tet repressor fused to DOZI. The small molecule ATc regulates the interaction between the repressor and the aptamer, which is found in the

3’ end of the untranslated region of the gene. In the absence of ATc, mRNAs are sequestered or targeted for degradation. In the presence of ATc, DOZI function is stabilized allowing for expression of genes to occur. Utilizing this system, we’ve characterized transgenic parasites expressing 1320-HA-Apatmer and 1320-BirA*-

Aptamer from the attB site and have shown that we can stringently control their 105 expression. We plan to knockout the endogenous copy of the PFF1320c gene in the

1320-HA-Apatamer parasites using the CRISPR-Cas9 system so that we can further explore the phenotype associated with depletion of PFF1320c.

Using the 1320-BirA*-Aptamer parasites, we executed biotinylation experiments where we incubated tightly synchronized 1320-BirA*-Aptamer parasites in the presence of ATc up to 24 hrs post invasion. Parasites where then washed with non-ATc containing media and allowed to incubate for an additional 2 hrs in the non-ATc media. Biotin was then added to the cultures and incubated for 4 hrs. Samples were then collected, purified, and analyzed by mass spectrometry. Over 25% of the proteins identified were specific to the 1320-BirA-Apatmer parasites versus the 3D7attB control in each replicate, with a total of 185 proteins detected at significant levels among the 1320-

BirA-Aptamer samples (Figure 3.20). Though we tried to adjust for each sample for comparable parasitemia between the 1320-BirA*-Aptamer and the controls, results were variable as we observed a greater number of proteins returned in the second run as compared to the first. A plausible explanation could lie within the synchronization of the parasites. As tightly synchronized parasites are within 12-18 hrs of the life cycle this is only an estimate, and it is possible by the second synchronization a majority of the parasites surpassed the 18 hr time point thus causing their loss from the first run.

PFF1320c ranked first among the 14 proteins found similar in both runs as expected, it biotinylated itself.

Actin in Plasmodium is expressed in two isoforms, ACTI and ACTII both of which are divergently different from their eukaryotic counterparts [126]. ACTI is directly 106 implicated in gliding motility, endocytic trafficking, gametocyte development, and cell- to-cell communication and is conversed between Plasmodium and other apicomplexan parasites. Marked by their highly dynamic, transient, and unstable nature ACTI filaments form very short polymers measuring only ~100 nm in length and are not readily detected using various microscope techniques [127, 128]. In vivo visualization of native actin-rich structures is only possible when cells are treated with an actin filament- stabilizing drug such as jasplakinolide [39, 127, 129]. Most work with ACT1 has relied on expression via heterologous systems to obtain enough purified protein for assaying.

When expressed in these conditions, P. falciparum ACT1 (PfACT1) is unable to fold into its native state and only forms long filaments when assayed in the presence of jasplakinolide or other F-actin binding drugs, thus supporting the observation that native apicomplexan actin forms unstable filaments preventing extended elongation

[130, 131]. In eukaryotic cells the chaperonin containing TCP-1 (CCT) complex folds actin into its mature native form. The TCP-1 complex is a 1-MDa-protein complex comprised of 8 different subunits arranged into 2 asymmetric rings [126]. Actin folding by the CCT complex is an ATP dependent, multistage process that is isoform and species specific, thus indicating a potential requirement for homologous protein folding machinery within the Plasmodium parasite cell. Olshina et al., reinforced this theory by showing that PfACT1 in a partially unfolded state disengages from GroEL and preferentially binds to the native Plasmodium CCT complex when prokaryotic in vitro translation lysate was incubated with Plasmodium cell extracts [126]. As we believe PFF1320c to be the MLC component of an unknown myosin motor complex, within the 14 proteins found similar 107 amongst runs in our biotinylation data set were 5 subunits from the CCT family of proteins. Interestingly, these proteins all have molecular weights ~60 KDa which correlates with an observed product of that size that may be made up of multiple products seen via western analysis when probed with StreptAvidin-HRP (Figure 3.19B).

The involvement of the T-complex in Plasmodium is not well studied. However, there are several hypotheses that we can draw to explain the involvement of PFF1320c with the CCT family of proteins.

First, in yeast it has been shown that all subunits of the CCT complex are essential and the individual subunits CCTα and CCTδ are essential in Tetrahymena [132].

As the complex is made up of eight individual subunits that disassociate to smaller assemblies and monomers at physiological potassium and ATP-concentrations [133] we can hypothesis that a) if the individual subunits found in our data set are indeed the cargo of PFF1320c it would explain our inability to knock out the endogenous PFF1320c gene and b) as the subunits of the CCT are cytoplasmic, we can also hypothesize that the

PFF1320c involved myosin motor complex assist in trafficking the subunits together to form the functional oligomeric complex.

Secondly, the expression levels of the individual CCT subunits are shown to be different from each other where CCTα and CCTδ are shown to be the lowest and most abundant subunits respectively in yeast [134]. In an oligomeric protein complex that requires all subunits in order to be functional, it would be assumed that individual expression of each subunit would be the equivalent. However, different expression levels between the subunits may be a result of an individual function of a particular 108 subunit or the result of subunits coming together in various combinations to accommodate a broad variety or substrates or substrates for folding [134]. Thus

PFF1320c may bind to these individual subunits to move them throughout the cytosol, which may explain the punctate staining observed in our immunofluorescence assays

(Figure 3.5).

Understandably identification by proximity-dependent labeling does not prove relationship. To confirm a relationship between PFF1320c and subunits of the CCT, we can generate tagged versions of the subunits and perform co-immunofluorescence and co-immunoprecipitation. Co-immunoprecipitation assays may also lead to identification of other components of this presumed myosin motor. We can also utilize conditional knockdown systems such as the glmS ribozyme with the CCT subunits to observe the effect on PFF1320c and the parasite. By characterizing the relationship of these T- complex proteins with PFF1320, we can hope to further elucidate the role of the protein and motor complex in within the Plasmodium parasite.

As previously mentioned there is a potential involvement of PFF1320c in hemoglobin uptake as Milani et al., has shown evidence that cytostomes use an actin myosin motor to transport hemoglobin to the digestive vacuole [122]. We identified one protein PF3D7_1020900, also known as ADP-Ribosylation Factor (Arf1), similar between both runs. The food vacuole membrane protein PfCRT is trafficked to the food vacuole membrane via the ER/Golgi [135]. Arf proteins are involved in the regulation of ER and

Golgi function, morphology, and of interest to us ER-to Golgi transport. It is plausible to suppose an interaction of Arf1 with PFF1320c. 109

In addition to determining proteins proximal to PFF1320c, we utilized Bio ID to explore proteins found in common between the MyoB-MTIPB motor complex. We hypothesized that utilization of this system would allow for the identification of putative substrates and interactors. Analysis identified 386 proteins that were common between both MyoB-BirA* and MTIPB-BirA* including both PfMyoB and PfMTIPB which is to be expected. Out of the 386, 21 proteins were of special interest to us as they contained functional properties consistent with or indicative of an involvement with a myosin motor complex. From this group we identified 10 proteins annotated with GO

Components encompassing that of a myosin complex, having motor activity, or binding to actin or calmodulin with molecular weights ranging from ~90-400 KDa. As consistently seen with many of the proteins in our overall data set 4/10 of these proteins were described as “conserved, unknown function”. Though these proteins are annotated in PlasmoDB as having properties of that of myosin complex it remains to be confirmed whether these proteins are actually involved in the stability or potential biological function of the MyoB-MTIPB complex. To investigate this we can attempt to knock out these proteins and observe the effects on both MyoB and MTIPB. The inability to knock out MyoB and MTIPB lends to the essential nature of the MyoB-MTIPB complex, however as several of these proteins have relatively large molecular weights indicative of that of a MHC as well as annotated GO Components and GO functions showing a relationship to myosin complexes, actin binding, and motor activity, it would be interesting to see if MTIPB would alternatively bind in situations where MyoB has 110 been knocked down and vice versa. Subsequently we can perform immunofluorescence and immunoprecipitation assays to confirm a direct interaction.

Of the 21 proteins that are of special interest to use we also identified 5 proteins with functional properties involving DNA replication belonging to the minichromsome maintenance complex (MCM) composed of MCM 2-7 that play a key role in the pre- replication complex. Though MCM is a heterohexameric complex, sub complexes of various configurations are detectable [136]. The Plasmodium genome encodes 8 MCM subunits (MCM2-MCM9) and encompasses several points in the life cycle where DNA replication occurs [137, 138]. Schizogeny is a process requiring multiple rounds of DNA replication. Studies show that PfMCM2, 6, and 7 forms a sub complex in the ring and schizonts stages and dissociate during the trophozoite stage. Although it’s premature to assign a role for the PfMyoB-MTIPB motor, due to its characteristically late expression during schizogeny one could speculate an involvement in supporting DNA replication and schizogeny.

Through these studies we have set out to characterize and elucidate the biological functions of proteins that make up Plasmodium molecular motors. To build on this work we plan to use the highly effective CRISPR-Cas9 genetic manipulation system to genetically disrupt the PFF1320c gene and monitor the effect on the Plasmodium parasite. Through use of Bio ID we’ve also identified several potential interactors as well as plausible roles in which PFF1320c as well as the MyoB-MTIPB motor may function.

We would like to further explore the involvement of PFF1320c in hemoglobin uptake as well as determine the possible role in aiding the T-complex proteins. These studies that 111 are intended for further investigation would be vital for unraveling aspects of these myosin motors that remain unknown.

112

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APPENDIX

122

Appendix I Primers

1320-5AV

5’-TATCACCTAGGTGATAAATGGAGGAAATAATTAATGAAGTG-3’

1320-3BS

5’- GAATCGTACGATTTAATATCCTGTTTAGAAATTCCTTATAGTC-3’

1320-5AP

5’-CATGCGGGCCCAATACGGCTACAGAAAATGAAAGTAGAAATAC-3’

1320-3AV

5’-TATCACCTAGGTAATTCTATATAAGATTAAATCTTATATGATAC3’

1320G-5KA

5’-GAAAAGGCGCCTGAACATCATGACAAGGAGGAAGATATTAATGC-3’

1320G-3NC

5’-GCAAACCATGGAAGCATTTGAGAAATCAATTTTAATTTCATG-3’

1320G-5AF

5’-GCTTAGCTTAAGTTTGACCACATGTATATATAACTATCTC-3’

1320-3SA

5’-GTTAGCCGCGGAAAATGTGTTATCTCCTATTCATGTTGCTGC-3’

1320-5AF

5’-CATGCCTTAAGAATACGGCTACAGAAAATGAAAGTAGAAATAC-3’

3HA-3AP

5’-CTAGTGGGCCCCGCCTCATCACTGAGCAGCGTAATCTGGAACG-3’

123

BIRA-3AP

5’- CTAGTGGGCCCTCATTATTGAGCTGCATAATCTGGCACATC-3’

1320-233F

5’-GATCAGGGTAATAAAGAAAAGGAAACAGATG-3’

1320-2686R

5’-CTTGTTAAAAAGAGAAGTCAATTCCACTTC-3’

1320-2910F

5’-GTTGGTTCAAAAATGTCGGTTGATGAAATG-3’

1320-4286R

5’-CTAATATACATGGAGAAACATCCTTAATATATTC-3’

PUF-1-2068F

5’-CATGTTTTGTAATTTATGGGATAGCG-3’

PUF-1-4035R

5’-TAAACCAATAGATAAAATTTGTAGAG-3

PFMYOB-5AP

5’-GAAGGGCCCGTGTATGAATATAATCCAACTTTGATTATATATATATTTATTTTAATTATG-3’

PFMYOB-3AV

5’-TATCACCTAGGTATTATATATTTTATTTATTAATAGTATAT-3’

PFMTIP-B-5AP

5’-GATAGGGCCCTAGTTGTAAGTTGACGAAGTGGTACAACGTATAC-3’

PFMTIP-B-3AV

5’-CGATCCTAGGGTTCTGCCTTTTTCTCTCTTACTACGTG

124

MYOC-5AF

5’-GAAGCTTAAGCATATGCTGTGCGTGTATCTAAAGCATGTAATGC-3’

MYOC-3BS

5’-GCAAGCGTACGTACAAAAGACCTGCGCCAGAAAACTATGTTTC-3’

MYOE-5XB

5’-CTTGTCTAGATGATATAAATGAGGAACAAAATAACATTGATG-3’

MYOE-3BS

5’-GCTACGTACGCAATGCTGGCGTTTTGAATACAAGGATG-3’

125

Appendix II: 1320-BirA* mass spectrometry data

1320-BirA*-Aptamer –Sample 1

Sequence MS/MS Protein IDs Fasta headers Peptides coverage Count [%] knob-associated histidine-rich PF3D7_0202000 protein 13 20.5 21 PF3D7_0211800 asparagine--tRNA ligase 6 14.8 6 PF3D7_0214000 T-complex protein 1 subunit theta 3 6.1 3 PF3D7_0303200 HAD superfamily protein, putative 5 5.2 6 PF3D7_0306800 T-complex protein 1 subunit beta 10 28.5 11 40S ribosomal protein S15A, PF3D7_0316800 putative 3 23.1 3 PF3D7_0322900 40S ribosomal protein S3A, putative 4 21 4 zinc finger Ran-binding domain- PF3D7_0408300 containing protein 2, putative 12 54.4 26 PF3D7_0417800 cdc2-related protein kinase 1 (CRK1) 13 17.9 13

PF3D7_0419600 ran binding protein 1, putative 5 20.4 7 PF3D7_0422400 40S ribosomal protein S19 (RPS19) 4 17.6 4 PF3D7_0503400 actin-depolymerizing factor 1 (ADF1) 3 43.4 3 nuclear protein localization protein PF3D7_0507700 4, putative (NPL4) 3 8.5 3 PF3D7_0517300 pre-mRNA-splicing factor (SR1) 7 32.6 8 S-adenosyl-L-homocysteine PF3D7_0520900 hydrolase (SAHH) 12 26.7 12

PF3D7_0527900 RNA helicase 5 8.3 5 PF3D7_0608700 chaperone, putative 360203(+) 3 7 2 PF3D7_0608800 ornithine aminotransferase (OAT) 5 17.9 5 PF3D7_0610400;PF3D7_ 0617900 histone H3 (H3) 4 43.4 4 transcription elongation factor SPT5, PF3D7_0610900 putative (SPT5) 8 6.1 8 conserved Plasmodium protein, PF3D7_0617200 unknown function 8 23.3 8 PF3D7_0617800 histone H2A 3 34.8 4 PF3D7_0624000 hexokinase 5 15.2 5 PF3D7_0626800 pyruvate kinase 14 47.9 20 126

PF3D7_0627200 myosin light chain, putative 10 89.9 21 PF3D7_0708400 heat shock protein 90 32 43.5 39

PF3D7_0711000 AAA family ATPase, CDC48 subfamily 10 11.9 11 sin3 associated polypeptide p18-like PF3D7_0711400 protein 8 7.8 10

PF3D7_0721600 40S ribosomal protein S5, putative 3 23.6 3

Plasmodium exported protein PF3D7_0731300 (PHISTb), unknown function 6 18.3 7 hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate PF3D7_0810800 synthase 5 8.6 4 PF3D7_0812400 karyopherin alpha 4 11.7 4

PF3D7_0812500 RNA-binding protein, putative 15 17.1 18 conserved Plasmodium protein, PF3D7_0813300 unknown function 5 20.6 5 PF3D7_0813900 40S ribosomal protein S16, putative 5 27.1 5 PF3D7_0818200 14-3-3 protein 3 22.9 3 PF3D7_0818900;PF3D7_ 0831700 heat shock protein 70 (HSP70 32 46.5 52 PF3D7_0823200 RNA-binding protein, putative 3 14.3 3 PF3D7_0826700 receptor for activated c kinase 6 23.5 6 replication protein A1, small PF3D7_0904800 fragment 6 16.5 7 PF3D7_0915400 6-phosphofructokinase 30 28.3 34 PF3D7_0917900 heat shock protein 70 9 17.3 8

PF3D7_0922200 S-adenosylmethionine synthetase 10 31.6 12 PF3D7_0922500 phosphoglycerate kinase 3 9.1 2 conserved Plasmodium protein, PF3D7_0924100 unknown function 8 13.3 8 PF3D7_0929200 RNA-binding protein, putative 3 11.1 3 cAMP-dependent protein kinase PF3D7_0934800 catalytic subunit 4 10.5 4 PF3D7_1004400 RNA-binding protein, putative 26 24.4 29 eukaryotic translation initiation PF3D7_1007900 factor 3 subunit D, putative 5 10.3 5 PF3D7_1008700 tubulin beta chain 3 9.4 4 PF3D7_1015900 enolase 6 19.3 6 PF3D7_1020900 ADP-ribosylation factor 4 32.6 4 PF3D7_1022400 serine/arginine-rich splicing factor 4 9 21.6 11

PF3D7_1026800 40S ribosomal protein S2 6 30.4 7 127

PF3D7_1027800 60S ribosomal protein L3 3 11.7 3 pre-mRNA-splicing factor ATP- dependent RNA helicase PRP22, PF3D7_1030100 putative 14 11.9 26 PF3D7_1034900 methionine--tRNA ligase 5 7.9 5 PF3D7_1104400 thioredoxin, putative 4 11.8 4 PF3D7_1105000 histone H4 3 34 3 PF3D7_1105100 histone H2B 6 33.3 6 PF3D7_1105400 40S ribosomal protein S4, putative 6 27.2 6 heat shock protein DnaJ homologue PF3D7_1108700 Pfj2 3 7.4 4 PF3D7_1110400 asparagine-rich antigen 7 5.5 10 GTP-binding nuclear protein PF3D7_1117700 RAN/TC4 3 15 4 splicing factor U2AF small subunit, PF3D7_1119300 putative 5 21.4 5 conserved Plasmodium protein, PF3D7_1120000 unknown function 4 13.5 7 PF3D7_1132200 T-complex protein 1 subunit alpha 3 10.5 3 PF3D7_1134100 protein disulfide isomerase 5 15.1 6 PF3D7_1136500.1;PF3D7 _1136500.2 casein kinase 1 (CK1) 4 12.4 4 PF3D7_1138500 protein phosphatase 2C 5 6.2 5 PF3D7_1142500 60S ribosomal protein L28 4 37.8 5

PF3D7_1144000 40S ribosomal protein S21 4 39 5 eukaryotic translation initiation PF3D7_1204300 factor 5A 4 31.1 7 glutathione peroxidase-like PF3D7_1212000 thioredoxin peroxidase 3 26.8 3 eukaryotic translation initiation PF3D7_1212700 factor 3 subunit A, putative 7 6.5 7 PF3D7_1222300 endoplasmin, putative 5 9.3 5 cAMP-dependent protein kinase PF3D7_1223100 regulatory subunit 3 9.1 3 PF3D7_1229500 T-complex protein 1 subunit gamma 7 14.8 7 pre-mRNA-splicing regulator, PF3D7_1230800 putative 8 17.3 10 PF3D7_1231100 ras-related protein Rab-2 3 16.9 3 pre-mRNA-splicing factor ATP- dependent RNA helicase PRP2, PF3D7_1231600 putative 16 22.6 20 ATP-dependent zinc PF3D7_1239700 metalloprotease FTSH 1 5 7 6

PF3D7_1246200 actin I 3 11.4 3 128

PF3D7_1302800 40S ribosomal protein S7, putative 4 33 4 conserved Plasmodium protein, PF3D7_1305900 unknown function 4 6.7 4 conserved Plasmodium protein, PF3D7_1314700 unknown function 3 5.8 3 PF3D7_1321700 splicing factor 1 12 15.5 16 PF3D7_1324900 L-lactate dehydrogenase 3 16.8 3 phosphoribosylpyrophosphate PF3D7_1325100 synthetase 8 27.2 9 PF3D7_1326300 RNA-binding protein, putative 10 23.7 13 PF3D7_1338300 elongation factor 1-gamma, putative 6 12.7 5 phosphoethanolamine N- PF3D7_1343000 methyltransferase 3 13.5 4 PF3D7_1349200 glutamate--tRNA ligase, putative 5 9.2 5 PF3D7_1357100;PF3D7_ 1357000 elongation factor 1-alpha 25 64.8 35 PF3D7_1357800 T-complex protein 1 subunit delta 11 27 12 PF3D7_1358800 40S ribosomal protein S15 4 21.2 5 conserved Plasmodium protein, PF3D7_1406200 unknown function 19 7.8 18 PF3D7_1408600 40S ribosomal protein S8e, putative 3 20.2 3 PF3D7_1412600 deoxyhypusine synthase 5 13.1 4 PF3D7_1424100 60S ribosomal protein L5, putative 3 13.6 3

PF3D7_1437900 HSP40, subfamily A, putative 3 14.9 3 PF3D7_1444800 fructose-bisphosphate aldolase 6 29.3 6 PF3D7_1445400 protein serine/threonine kinase-1 5 6.4 6 ATP-dependent RNA helicase DDX5, PF3D7_1445900 putative 5 13.1 4 PF3D7_1447000 40S ribosomal protein S5 3 11.4 3

PF3D7_1451100 elongation factor 2 14 25.4 18 U1 snRNA associated protein, PF3D7_1452700 putative 4 14 4 PF3D7_1457300 co-chaperone p23 3 8 3 glyceraldehyde-3-phosphate PF3D7_1462800 dehydrogenase 14 59.6 15 PF3D7_1465900 40S ribosomal protein S3 3 15.8 3 PF3D7_1468700 eukaryotic initiation factor 4A 8 31.2 10 PF3D7_1471100 exported protein 2 3 9.8 3

3D7attB- Sample 1 Sequence Protein IDs Fasta headers Peptides MS/MS coverage [%] Count PF3D7_0812500 RNA-binding protein, putative 24 23.6 18 129

pre-mRNA-splicing factor ATP-dependent PF3D7_1030100 RNA helicase PRP22, putative (PRP22) 23 18.9 26 PF3D7_0915400 6-phosphofructokinase (PFK9) 21 22.1 34 PF3D7_0818900;PF 3D7_0831700 heat shock protein 70 (HSP70 19 35.9 52 PF3D7_1004400 RNA-binding protein, putative 16 15.3 29 PF3D7_0408300 zinc finger, RAN binding protein, putative 15 60.8 26 conserved Plasmodium protein, unknown PF3D7_1406200 function 14 6.3 18 pre-mRNA-splicing factor ATP-dependent PF3D7_1231600 RNA helicase PRP2, putative (PRP2) 13 16.7 20 PF3D7_1451100 product=elongation factor 2 12 27 18 PF3D7_0626800 product=pyruvate kinase (PyrK) 11 43.4 20 glyceraldehyde-3-phosphate PF3D7_1462800 dehydrogenase (GAPDH) 10 37.4 15 knob-associated histidine-rich protein PF3D7_0202000 (KAHRP) 10 18.3 21 sin3 associated polypeptide p18-like PF3D7_0711400 protein 10 9.8 10 PF3D7_1357100;PF 3D7_1357000 elongation factor 1-alpha 9 29.1 35 PF3D7_1321700 splicing factor 1 (SF1) 9 11.9 16 PF3D7_0303200 HAD superfamily protein, putative 9 9.6 6 eukaryotic translation initiation factor 3 PF3D7_1212700 subunit 10, putative 9 8.8 7 PF3D7_1110400 asparagine-rich antigen 9 6.1 10 PF3D7_0517300 pre-mRNA-splicing factor (SR1) 8 32.6 8 PF3D7_1326300 RNA-binding protein, putative 8 21.4 13 PF3D7_1445400 protein serine/threonine kinase-1 (CLK1) 8 10.1 6 PF3D7_0922200 S-adenosylmethionine synthetase (SAMS) 7 21.4 12 conserved Plasmodium protein, unknown PF3D7_1230800 function 7 13.4 10 conserved Plasmodium protein, unknown PF3D7_0924100 function 7 13.2 8 splicing factor U2AF small subunit, PF3D7_1119300 putative (U2AF1) 6 29.3 5 PF3D7_1104400 conserved protein, unknown function 6 14.9 4 PF3D7_0917900 heat shock protein 70 (HSP70-2) 6 11.2 8 PF3D7_0417800 cdc2-related protein kinase 1 (CRK1) 6 8.3 13 AAA family ATPase, CDC48 subfamily PF3D7_0711000 (Cdc48) 6 5.9 11 transcription elongation factor SPT5, PF3D7_0610900 putative (SPT5) 6 5.8 8 PF3D7_1105100 histone H2B (H2B) 5 36.8 6 PF3D7_1325100 phosphoribosylpyrophosphate synthetase 5 16.5 9 conserved Plasmodium protein, unknown PF3D7_1120000 function 5 15.8 7 serine/arginine-rich splicing factor 4 PF3D7_1022400 (SRSF4) 5 15.4 11 130

S-adenosyl-L-homocysteine hydrolase PF3D7_0520900 (SAHH) 5 9.6 12 PF3D7_0211800 asparagine--tRNA ligase (AsnRS) 5 9.3 6 PF3D7_1307800 fork head domain protein, putative 5 8.9 3 eukaryotic translation initiation factor 3 PF3D7_1007900 subunit 7, putative 5 6.8 5 PF3D7_1142500 60S ribosomal protein L28 (RPL28) 4 35.4 5 PF3D7_0422400 40S ribosomal protein S19 (RPS19) 4 24.1 4 PF3D7_1026800 40S ribosomal protein S2 (RPS2) 4 18.6 7 PF3D7_1424100 60S ribosomal protein L5, putative 4 16.3 3 Plasmodium exported protein (PHISTb), PF3D7_0731300 unknown function (PfG174) 4 15.5 7 conserved Plasmodium protein, unknown PF3D7_0617200 function 4 14.6 8 PF3D7_0608800 ornithine aminotransferase (OAT) 4 13.5 5 ATP-dependent RNA helicase DDX5, PF3D7_1445900 putative (DDX5) 4 10.8 4 PF3D7_1108700 heat shock protein DnaJ homologue Pfj2 4 9.3 4 ATP-dependent zinc metalloprotease FTSH PF3D7_1239700 1 (FTSH1) 4 8.2 6 PF3D7_0527900 RNA helicase 1 4 6.3 5 conserved Plasmodium protein, unknown PF3D7_0813000 function 4 6.3 1 histone H2A (H2A) | PF3D7_0617800 location=Pf3D7_06_v3:748416-748814(-) 3 34.8 4 PF3D7_1144000 40S ribosomal protein S21 (RPS21) 3 25.6 5 PF3D7_1358800 40S ribosomal protein S15 (RPS15) 3 20.5 5 PF3D7_0610400;PF 3D7_0617900 histone H3 (H3) 3 19.9 4 PF3D7_1105400 40S ribosomal protein S4, putative 3 12.6 6 PF3D7_1324900 L-lactate dehydrogenase (LDH) 3 12 3 PF3D7_1468700 eukaryotic initiation factor 4A (eIF4A) 3 11.6 10 Plasmodium exported protein (PHISTb), PF3D7_0532300 unknown function | 3 10.2 0 PF3D7_1471100 exported protein 2 (EXP2) 3 9.8 3 serine/arginine-rich splicing factor 12 PF3D7_0503300 (SRSF12) 3 7.1 3 PF3D7_0507500 subtilisin-like protease 1 (SUB1) 3 6.4 0 conserved Plasmodium protein, unknown PF3D7_1305900 function 3 6.3 4 PF3D7_1222300 endoplasmin, putative (GRP94) 3 6.1 5

1320-BirA*-Aptamer Sample 2 Sequence MS/MS Protein IDs Fasta headers Peptides coverage [%] Count PF3D7_0627200 myosin light chain, putative 11 92.1 29 glyceraldehyde-3-phosphate PF3D7_1462800 dehydrogenase (GAPDH) | 25 81.3 40 131

PF3D7_1144000 40S ribosomal protein S21 (RPS21) 8 79.3 10 PF3D7_1357100;PF 3D7_1357000 elongation factor 1-alpha 39 78.3 76 PF3D7_0826700 receptor for activated c kinase (RACK) 20 77.1 27 PF3D7_0721600 40S ribosomal protein S5, putative 12 74.4 16 PF3D7_0516200 40S ribosomal protein S11 8 74.2 11 PF3D7_1006800 RNA-binding protein, putative 21 72.8 46 PF3D7_0719700 40S ribosomal protein S10, putative 12 72.3 20 PF3D7_0322900 40S ribosomal protein S3A, putative 27 72.1 38 eukaryotic translation initiation factor 3 PF3D7_1007900 subunit 7, putative 38 69.1 50 PF3D7_1325100 phosphoribosylpyrophosphate synthetase 26 68 31 PF3D7_1331800 60S ribosomal protein L23, putative 7 67.6 12 PF3D7_0818900;PF 3D7_0831700 heat shock protein 70 (HSP70) 56 67.1 105 PF3D7_0929200 RNA-binding protein, putative 16 66.8 26 PF3D7_1019400 60S ribosomal protein L30e, putative 7 66.7 10 PF3D7_0422400 40S ribosomal protein S19 (RPS19) 15 65.9 22 PF3D7_1437900 HSP40, subfamily A, putative 23 65.1 29 PF3D7_1421200 40S ribosomal protein S25 (RPS25) 11 64.8 14 PF3D7_1142500 60S ribosomal protein L28 (RPL28) 11 64.6 15 PF3D7_1134100 protein disulfide isomerase (PDI-11) 25 64.1 43 Plasmodium exported protein (PHISTb), PF3D7_0731300 unknown function (PfG174) 26 64 53 PF3D7_1105400 40S ribosomal protein S4, putative 18 63.6 26 PF3D7_1426000 60S ribosomal protein L21 (RPL21) 14 63.4 21 PF3D7_1447000 40S ribosomal protein S5 16 62.5 25 PF3D7_1341200 60S ribosomal protein L18, putative 15 62 22 PF3D7_0708400 heat shock protein 90 (HSP90) 54 61.7 89 PF3D7_0408300 zinc finger, RAN binding protein, putative 19 61.3 35 PF3D7_1358800 40S ribosomal protein S15 (RPS15) 12 60.9 19 PF3D7_1202900 high mobility group protein B1 (HMGB1) 7 60.8 9 conserved Plasmodium protein, unknown PF3D7_1230800 function 38 60.1 68 PF3D7_1011800 PRE-binding protein (PREBP) 92 59.9 162 PF3D7_0608800 ornithine aminotransferase (OAT) 23 59.7 31 PF3D7_1460700 60S ribosomal protein L27 (RPL27) 14 59.6 21 PF3D7_1126200 40S ribosomal protein S18, putative 8 59.6 13 PF3D7_1338300 elongation factor 1-gamma, putative 20 59.1 26 PF3D7_1414300 60S ribosomal protein L10, putative 14 58.9 24 PF3D7_1424100 60S ribosomal protein L5, putative 28 58.8 50 PF3D7_1015900 enolase (ENO) 17 58.5 22 PF3D7_0915400 6-phosphofructokinase (PFK9) 66 58.1 101 PF3D7_0626800 pyruvate kinase (PyrK) 17 58.1 29 AAA family ATPase, CDC48 subfamily PF3D7_0711000 (Cdc48) 71 57.9 105 132

PF3D7_1468700 eukaryotic initiation factor 4A (eIF4A) 16 57.8 23 PF3D7_0516900 60S ribosomal protein L2 (RPL2) 15 57.7 28 PF3D7_0818200 14-3-3 protein (14-3-3I) 9 57.6 11 PF3D7_0617800 histone H2A (H2A) 7 57.6 11 PF3D7_1016300;PF 3D7_0937000 glycophorin binding protein (GBP) 6 57.6 8 PF3D7_0316800 40S ribosomal protein S15A, putative 10 56.9 12 PF3D7_1108700 heat shock protein DnaJ homologue Pfj2 34 56.7 56 PF3D7_0817900 high mobility group protein B2 (HMGB2) 6 56.6 7 PF3D7_0306800 T-complex protein beta subunit, putative 21 56.5 30 PF3D7_1323100 60S ribosomal protein L6, putative 7 55.8 10 40S ribosomal protein S11, putative PF3D7_0317600 (RPS11) 10 55.3 18 PF3D7_1002400.1; PF3D7_1002400.2 RNA-binding protein, putative 24 54.8 38 PF3D7_1357800 TCP-1/cpn60 chaperonin family, putative 19 54.6 22 PF3D7_1465900 40S ribosomal protein S3 12 54.3 16 PF3D7_0503400 actin-depolymerizing factor 1 (ADF1) 4 54.1 6 PF3D7_1027800 60S ribosomal protein L3 (RPL3) 23 53.9 33 multiprotein bridging factor type 1, PF3D7_1128200 putative (MBF1) 8 53.7 8 PF3D7_0415500 nuclear cap-binding protein, putative 14 53 19 PF3D7_1302800 40S ribosomal protein S7, putative 10 52.6 13 PF3D7_1136300 tudor staphylococcal nuclease (TSN) 43 52.5 51 PF3D7_0517000 60S ribosomal protein L12, putative 9 52.1 9 PF3D7_0823200 RNA-binding protein, putative 15 51.7 23 PF3D7_1104400 conserved protein, unknown function 21 51.4 31 PF3D7_0517300 pre-mRNA-splicing factor (SR1) 28 51.3 42 PF3D7_1108400 casein kinase 2, alpha subunit (CK2alpha) 13 51.3 16 PF3D7_0903900 60S ribosomal protein L32 (RPL32) 8 51.1 13 PF3D7_0507100 60S ribosomal protein L4 (RPL4) 26 50.9 37 PF3D7_1472000 pre-mRNA-splicing factor ISY1, putative 6 50.7 10 PF3D7_0705700 40S ribosomal protein S29, putative 3 50 3 PF3D7_1338200 60S ribosomal protein L6-2, putative 9 49.8 12 PF3D7_1242700 40S ribosomal protein S17, putative 6 49.6 7 PF3D7_1109900 60S ribosomal protein L36 (RPL36) 8 49.1 13 PF3D7_1004400 RNA-binding protein, putative 65 49 92 PF3D7_0922200 S-adenosylmethionine synthetase (SAMS) 16 49 24 PF3D7_0917900 heat shock protein 70 (HSP70-2) 26 48.9 36 PF3D7_1026800 40S ribosomal protein S2 (RPS2) 13 48.7 18 ATP-dependent RNA helicase DDX6 PF3D7_0320800 (DOZI) 11 48.7 10 PF3D7_1243600 translation initiation factor SUI1, putative 5 48.7 5 PF3D7_0527900 RNA helicase 1 35 48.6 50 PF3D7_1451100 elongation factor 2 29 48.1 40 PF3D7_1360900 polyadenylate-binding protein, putative 13 46.8 24 133

PF3D7_0922500 phosphoglycerate kinase (PGK) 13 46.4 15 PF3D7_1444800 fructose-bisphosphate aldolase (FBPA) 11 46.3 14 PF3D7_0409400 heat shock protein 40 (DnaJ) 23 46.1 32 PF3D7_0624000 hexokinase (HK) 14 46 18 PF3D7_0307200 60S ribosomal protein L7, putative 14 45.9 18 PF3D7_0813900 40S ribosomal protein S16, putative 7 45.8 9 ATP-dependent RNA helicase DDX5, PF3D7_1445900 putative (DDX5) 17 45.7 23 PF3D7_0610400;PF 3D7_0617900 histone H3 (H3) 4 45.6 5 PF3D7_1408600 40S ribosomal protein S8e, putative 11 45.4 15 PF3D7_0618300 60S ribosomal protein L27a, putative 5 45.3 7 PF3D7_0814000 60S ribosomal protein L13-2, putative 8 45.1 13 PF3D7_0814200 DNA/RNA-binding protein Alba 1 (ALBA1) 12 44.8 18 splicing factor U2AF small subunit, PF3D7_1119300 putative (U2AF1) 12 44.6 15 PF3D7_1003500 40S ribosomal protein S20e, putative 6 44.1 7 PF3D7_0217800 40S ribosomal protein S26 (RPS26) 5 43.9 6 Plasmodium exported protein (PHISTb), PF3D7_0424600 unknown function 10 43.7 12 PF3D7_1130200 60S ribosomal protein P0 (PfP0) 10 43.7 11 PF3D7_1238100 calcyclin binding protein, putative 8 43.4 10 PF3D7_1407100 1407100 | fibrillarin, putative (NOP1) 13 43.1 19 GTP-binding nuclear protein RAN/TC4 PF3D7_1117700 (RAN) 9 43 12 PF3D7_0312800 60S ribosomal protein L26, putative 7 42.9 10 PF3D7_0502100 HCNGP-like protein 8 42.7 9 PF3D7_1412600 deoxyhypusine synthase (DHS) 18 42.5 22 PF3D7_1008700 tubulin beta chain 10 42.5 12 Plasmodium exported protein (PHISTc), PF3D7_1148700 unknown function (GEXP12) 10 42.2 16 PF3D7_1424400 60S ribosomal protein L7-3, putative 14 42 17 PF3D7_1452700 U1 snRNA associated protein, putative 11 42 18 PF3D7_1105100 histone H2B (H2B) 9 41.9 18 phosphoglycerate mutase, putative PF3D7_1120100 (PGM1) 8 41.6 8 PF3D7_0417800 cdc2-related protein kinase 1 (CRK1) 22 41.5 33 glutathione peroxidase-like thioredoxin PF3D7_1212000 peroxidase (TPx(Gl)) 7 41.5 8 pre-mRNA-splicing factor ATP-dependent PF3D7_1231600 RNA helicase PRP2, putative (PRP2) 32 41 38 PF3D7_1134000 heat shock protein 70 (HSP70-3) 26 40.9 31 eukaryotic translation initiation factor 5A PF3D7_1204300 (EIF5A) 7 40.4 11 PF3D7_0406100 vacuolar ATP synthase subunit b 10 40.1 9 Plasmodium exported protein (PHISTc), PF3D7_0801000 unknown function 29 40 39 134

nuclear preribosomal assembly protein, PF3D7_1124800 putative 9 39.8 10 eukaryotic translation initiation factor 3 PF3D7_1212700 subunit 10, putative 54 38.6 67 PF3D7_1317800 40S ribosomal protein S19 (RPS19) 6 38.6 12 PF3D7_1438900 thioredoxin peroxidase 1 (Trx-Px1) 4 38.5 6 conserved Plasmodium protein, unknown PF3D7_0617200 function 21 38.3 28 eukaryotic translation initiation factor 2 PF3D7_1010600 beta subunit, putative 7 38.3 7 PF3D7_1345100 thioredoxin 2 (TRX2) 6 38.2 7 PF3D7_1341300 60S ribosomal protein L18-2, putative 6 38 9 PF3D7_1351400 60S ribosomal protein L17, putative 7 37.9 10 PF3D7_1308300 40S ribosomal protein S27 (RPS27) 3 37.8 3 PF3D7_1020900 ADP-ribosylation factor (ARF1) 4 37.6 4 PF3D7_0730900 EMP1-trafficking protein (PTP4) 23 37.3 39 PF3D7_1015600 heat shock protein 60 (HSP60) 14 37.1 15 PF3D7_1431700 60S ribosomal protein L14, putative 6 37 7 PF3D7_1222300 endoplasmin, putative (GRP94) 23 36.7 21 eukaryotic translation initiation factor, PF3D7_0517700 putative 16 36.6 16 PF3D7_1136500.1; PF3D7_1136500.2 casein kinase 1 (CK1) 11 36.5 11 PF3D7_0210100.1 60S ribosomal protein L37ae, putative 4 36.5 3 PF3D7_1342000 40S ribosomal protein S6 14 36.3 17 eukaryotic translation initiation factor PF3D7_1438000 eIF2A, putative 16 36.2 19 replication protein A1, small fragment PF3D7_0904800 (RPA1) 12 36.2 13 PF3D7_0812500 RNA-binding protein, putative 43 36.1 61 PF3D7_0923900 RNA-binding protein, putative 4 36.1 4 PF3D7_0823800 DnaJ protein, putative 17 36 20 PF3D7_1246200 actin I (ACT1) 9 35.4 12 serine/arginine-rich splicing factor 4 PF3D7_1022400 (SRSF4) 32 35.1 52 PF3D7_1426100 basic transcription factor 3b, putative 6 35.1 5 PF3D7_1362200 RuvB-like helicase 3 (RUVB3) 10 34.8 10 PF3D7_0706400 60S ribosomal protein L37 (RPL37) 5 34.8 6 PF3D7_0722400 GTP-binding protein, putative 7 34.4 8 PF3D7_0415900 60S ribosomal protein L15, putative 6 34.1 7 PF3D7_0304400 60S ribosomal protein L44 (RPL44) 7 33.7 9 conserved Plasmodium protein, unknown PF3D7_1445700 function 8 33 13 PF3D7_0320900 histone H2A variant, putative (H2A.Z) 3 32.9 4 PF3D7_1365900;PF 3D7_1211800 1 ubiquitin-60S ribosomal protein L40 4 32.8 5 PF3D7_0503800 60S ribosomal protein L31 (RPL31) 4 32.5 6 135

conserved Plasmodium protein, unknown PF3D7_1305900 function 16 32.4 23 conserved Plasmodium protein, unknown PF3D7_1459400 function 8 32.1 9 hypoxanthine-guanine PF3D7_1012400 phosphoribosyltransferase (HGPRT) 3 32 3 PF3D7_1008800 nucleolar protein 5, putative (NOP5) 11 31.8 12 PF3D7_1471100 exported protein 2 (EXP2) 8 31.7 15 PF3D7_1323400 60S ribosomal protein L23 (RPL23) 8 31.6 18 PF3D7_0319500 RNA-binding protein, putative 8 31.4 11 PF3D7_1004000 60S ribosomal protein L13, putative 6 31.2 7 Plasmodium exported protein, unknown PF3D7_1149400 function 5 30.6 6 PF3D7_0213100;PF 3D7_0501100.2;PF 3D7_0501100.1 heat shock protein 40, putative 7 30.2 8 knob-associated histidine-rich protein PF3D7_0202000 (KAHRP) 26 30.1 51 conserved Plasmodium protein, unknown PF3D7_1419700 function 12 30.1 20 U1 small nuclear ribonucleoprotein, PF3D7_1367100 putative 14 30 21 PF3D7_0303200 HAD superfamily protein, putative 24 29.6 36 Plasmodium exported protein (PHISTb), PF3D7_1476200 unknown function 10 29.5 14 PF3D7_0629200 DnaJ protein, putative 8 29.5 10 debranching enzyme-associated PF3D7_1220400 ribonuclease, putative (DRN1) 15 29.3 17 PF3D7_1136800 DnaJ protein, putative 7 29.3 6 ATP-dependent RNA helicase UAP56 PF3D7_0209800 (UAP56) 7 29.3 8 conserved Plasmodium protein, unknown PF3D7_0813300 function 11 29.2 17 eukaryotic translation initiation factor 3 PF3D7_0918300 subunit 5, putative 5 29.1 4 nucleolar rRNA processing protein, PF3D7_0722600 putative 12 28.9 14 PF3D7_1130100 60S ribosomal protein L38 (RPL38) 3 28.7 4 membrane associated histidine-rich PF3D7_1370300 protein (MAHRP1) 3 28.5 2 conserved Plasmodium protein, unknown PF3D7_0924100 function 22 28.4 32 conserved Plasmodium protein, unknown PF3D7_1330800 function 12 28.4 22 RNA-binding protein musashi, putative PF3D7_0916700 (HoMu) 8 28.1 11 PF3D7_1119200 conserved protein, unknown function 4 28 5 serine/arginine-rich splicing factor 12 PF3D7_0503300 (SRSF12) 13 27.9 25 PF3D7_1352500 thioredoxin-related protein, putative 5 27.9 7 136

PF3D7_0903700;PF 3D7_0422300 alpha tubulin 1 7 27.6 8 PF3D7_0306900 40S ribosomal protein S23, putative 3 27.6 3 PF3D7_0605100 RNA-binding protein, putative 17 27.4 22 vesicle-associated membrane protein, PF3D7_1439800 putative 6 27.4 9 eukaryotic translation initiation factor 3 PF3D7_1206200 subunit 8, putative 21 27.2 26 PF3D7_0812400 karyopherin alpha (KARalpha) 8 27 11 PF3D7_1236100 clustered-asparagine-rich protein 6 27 8 PF3D7_1203700 nucleosome assembly protein (NAPL) 7 26.8 9 PF3D7_0827900 protein disulfide isomerase (PDI8) 6 26.7 6 PF3D7_1360100 RNA-binding protein, putative 3 26.7 3 PF3D7_1326300 RNA-binding protein, putative 15 26.6 36 PF3D7_0211800 asparagine--tRNA ligase (AsnRS) 9 26.6 12 PF3D7_0821700 60S ribosomal protein L22, putative 3 26.6 3 PF3D7_0813600 translation initiation factor SUI1, putative 8 26.5 17 PF3D7_1034900 methionine--tRNA ligase, putative 14 26.4 15 conserved Plasmodium protein, unknown PF3D7_1120000 function 12 26.3 21 box C/D snoRNP rRNA 2-O-methylation PF3D7_1118500 factor, putative 8 26.3 11 PF3D7_1227100 DNA helicase 60 (DH60) 12 26.1 12 U4/U6 snRNA-associated-splicing factor, PF3D7_0920900 putative (PRP24) 14 26 15 PF3D7_1472900 dihydroorotase, putative 6 26 7 PF3D7_0710600 60S ribosomal protein L34 (RPL34) 6 26 6 PF3D7_0714000 histone H2B variant (H2B.Z) 3 26 4 PF3D7_1309100 60S ribosomal protein L24, putative 6 25.9 7 PF3D7_1307800 fork head domain protein, putative 14 25.8 17 PF3D7_1315400 zinc finger (CCCH type) protein, putative 9 25.8 14 GrpE protein homolog, mitochondrial, PF3D7_1124700 putative (MGE1) 5 25.6 6 S-adenosyl-L-homocysteine hydrolase PF3D7_0520900 (SAHH) 10 25.3 10 ADP/ATP transporter on adenylate PF3D7_1037300 translocase (ADT) 6 25.2 9 PF3D7_1224000 GTP cyclohydrolase 6 25.2 6 translation initiation factor eIF-1A, PF3D7_1143400 putative 4 25.2 4 PF3D7_0315100 translation initiation factor 4E (eIF4E) 5 25.1 6 PF3D7_1311400 AP-1 complex subunit mu, putative 7 24.9 8 PF3D7_0110400 DNA-directed RNA polymerase 2, putative 5 24.9 7 conserved Plasmodium protein, unknown PF3D7_0722000 function 4 24.9 5 PF3D7_1324900 L-lactate dehydrogenase (LDH) 4 24.7 4 eukaryotic translation initiation factor, PF3D7_0815600 putative 4 24.6 4 137

conserved Plasmodium protein, unknown PF3D7_1025300 function 19 24 23 conserved Plasmodium protein, unknown PF3D7_0519700 function 4 24 4 conserved Plasmodium protein, unknown PF3D7_0721100 function 4 23.8 3 PF3D7_1118200 heat shock protein 90, putative 14 23.7 15 PF3D7_1334500 MSP7-like protein (MSRP6) 13 23.6 17 PF3D7_0614500 60S ribosomal protein L19 (RPL19) 6 23.6 7 ATP-dependent zinc metalloprotease FTSH PF3D7_1239700 1 (FTSH1) 13 23.5 20 haloacid dehalogenase-like hydrolase PF3D7_1033400 (HAD1) 3 23.3 3 1 T-complex protein 1, gamma subunit, PF3D7_1229500 putative 8 23.1 9 ubiquitin-40S ribosomal protein S27a, PF3D7_1402500 putative 3 22.8 2 conserved Plasmodium protein, unknown PF3D7_1406200 function 57 22.7 75 PF3D7_0609200 citrate synthase-like protein, putative 8 22.4 9 PF3D7_1321700 splicing factor 1 (SF1) 19 22.3 38 PF3D7_1220900 heterochromatin protein 1 (HP1) 3 22.2 5 PF3D7_0810600 RNA helicase, putative 12 22 13 conserved Plasmodium protein, unknown PF3D7_1036900 function 36 21.8 44 sin3 associated polypeptide p18-like PF3D7_0711400 protein 20 21.8 27 phosphoethanolamine N- PF3D7_1343000 methyltransferase (PMT) 4 21.8 5 PF3D7_0607000 translation initiation factor IF-2, putative 12 21.7 13 PF3D7_0520000 40S ribosomal protein S9, putative 5 21.7 8 chaperone, putative | PF3D7_0608700 location=Pf3D7_06_v3:358391-360203(+) 9 21.4 10 pre-mRNA-splicing factor ATP-dependent PF3D7_1030100 RNA helicase PRP22, putative (PRP22) 27 21.2 52 Plasmodium exported protein (PHISTa), PF3D7_0402000 unknown function 13 21 37 conserved Plasmodium protein, unknown PF3D7_0921900 function 4 21 5 large subunit rRNA processing protein, PF3D7_1405800 putative 12 20.7 14 RNA binding protein Bruno, putative PF3D7_1409800 (HoBo) 8 20.6 9 Plasmodium exported protein, unknown PF3D7_0831400 function 6 20.5 6 autophagy-related protein 18, putative PF3D7_1012900 (ATG18) 4 20 5 PF3D7_1238800 acyl-CoA synthetase (ACS11) 10 19.9 12 conserved Plasmodium protein, unknown PF3D7_1026000 function 6 19.8 7 138

PF3D7_0419600 ran binding protein 1, putative 5 19.6 3 PF3D7_1415300 RNA-binding protein Nova-1, putative 4 19.6 4 conserved Plasmodium protein, unknown PF3D7_1105800 function 5 19.5 6 PF3D7_1110400 asparagine-rich antigen 33 19.4 49 transcription elongation factor SPT5, PF3D7_0610900 putative (SPT5) 24 19.4 29 PF3D7_1346300 DNA/RNA-binding protein Alba 2 (ALBA2) 3 19.4 4 1 isocitrate dehydrogenase (NADP), PF3D7_1345700 mitochondrial precursor (IDH) 6 19.2 7 PF3D7_0719600 60S ribosomal protein L11a, putative 3 19.1 5 small subunit rRNA processing factor, PF3D7_1207100 putative 19 18.9 30 PF3D7_0707400 AAA family ATPase, putative 9 18.9 10 PF3D7_0420400 ribosome-recycling factor (RRF2) 3 18.8 4 conserved Plasmodium protein, unknown PF3D7_0403200 function 14 18.6 17 conserved Plasmodium protein, unknown PF3D7_0801500 function 7 18.6 8 PF3D7_0630900 ATP-dependent RNA helicase HAS1 (HAS1) 7 18.6 7 phosphoacetylglucosamine mutase, PF3D7_1130000 putative (PAGM) 11 18.4 13 PF3D7_1349200 glutamate--tRNA ligase, putative 10 18.2 12 M1-family alanyl aminopeptidase PF3D7_1311800 (M1AAP) 11 17.9 11 conserved Plasmodium protein, unknown PF3D7_0723900 function 21 17.8 24 conserved Plasmodium protein, unknown PF3D7_0214400 function 6 17.8 6 PF3D7_0913200 elongation factor 1-beta (EF-1beta) 6 17.8 7 PF3D7_0501300 skeleton-binding protein 1 (SBP1) 3 17.8 5 PF3D7_0922100 ubiquitin-like protein, putative 22 17.7 26 signal recognition particle receptor, beta PF3D7_1246800 subunit (SRPR-beta) 3 17.6 3 conserved Plasmodium protein, unknown PF3D7_1423700 function 28 17.5 35 PF3D7_1212900 bromodomain protein, putative 14 17.5 16 polyadenylate-binding protein, putative PF3D7_1224300 (PABP) 10 17.5 10 pre-mRNA-splicing factor 38B, putative PF3D7_1407300 (PRP38B) 8 17.1 7 conserved Plasmodium protein, unknown PF3D7_1409600 function 7 17.1 8 PF3D7_1453700 co-chaperone p23 (P23) 3 17.1 5 eukaryotic translation initiation factor 3 PF3D7_0716800 37.28 kDa subunit, putative 3 17.1 3 T-complex protein 1 epsilon subunit, PF3D7_0320300 putative 6 17 6 PF3D7_1358100 small subunit rRNA processing factor, 16 16.9 20 139

putative pyridoxine biosynthesis protein PDX1 PF3D7_0621200 (PDX1) 3 16.6 5 PF3D7_1005500 regulator of nonsense transcripts, putative 15 16.4 18 high molecular weight rhoptry protein 2 PF3D7_0929400 (RhopH2) 13 16.4 14 PF3D7_1132200 TCP-1/cpn60 chaperonin family, putative 5 16.2 5 PF3D7_1139900 conserved protein, unknown function 3 16.2 3 ring-infected erythrocyte surface antigen PF3D7_0102200 (RESA) 17 15.9 24 PF3D7_1246600 pre-mRNA-splicing factor, putative 4 15.6 6 PF3D7_0310300 phosphoglycerate mutase, putative 13 15.5 15 PF3D7_1410200 cytidine triphosphate synthetase 10 15.5 11 PF3D7_0102900 aspartate--tRNA ligase 5 15.5 5 PF3D7_0311100 pre-mRNA splicing factor, putative 12 15.3 15 PF3D7_0818000 conserved protein, unknown function 3 15.3 3 PF3D7_0414000 chromosome associated protein, putative 11 15 11 DEAD/DEAH box ATP-dependent RNA PF3D7_1251500 helicase, putative 8 15 10 conserved Plasmodium protein, unknown PF3D7_0521000 function 10 14.9 12 methionine aminopeptidase 1b, putative PF3D7_1015300 (METAP1b) 5 14.7 6 PF3D7_1333400 conserved protein, unknown function 3 14.7 3 PF3D7_1445400 protein serine/threonine kinase-1 (CLK1) 12 14.6 13 PF3D7_1434600 1methionine aminopeptidase 2 (METAP2) 8 14.5 11 ATP-dependent zinc metalloprotease PF3D7_1464900 FTSH, putative 5 14.4 5 conserved Plasmodium protein (10b PF3D7_1021900 antigen), unknown function 33 14.2 44 PF3D7_0624600 SNF2 helicase, putative (ISWI) 27 14.1 31 mature parasite-infected erythrocyte surface antigen,erythrocyte membrane PF3D7_0500800 protein 2 (MESA) 18 14 27 eukaryotic translation initiation factor 3 PF3D7_0612100 subunit L, putative 7 14 7 PF3D7_1318800 translocation protein SEC63 (SEC63) 6 14 7 conserved Plasmodium protein, unknown PF3D7_0813000 function 15 13.9 20 PF3D7_0519800 conserved protein, unknown function 6 13.9 6 conserved Plasmodium protein, unknown PF3D7_1317600 function 5 13.9 6 ATP-dependent RNA helicase DDX23, PF3D7_0518500 putative (DDX23 10 13.6 12 conserved Plasmodium protein, unknown PF3D7_1147300 function 12 13.2 15 PF3D7_0106300 calcium-transporting ATPase (ATP6) 9 13.2 9 pre-mRNA-splicing factor 38A, putative PF3D7_1132600 (PRP38A) 5 13.2 5 140

eukaryotic translation initiation factor 3, PF3D7_0528200 subunit 6, putative | 4 13.2 4 conserved Plasmodium protein, unknown PF3D7_0706500 function 18 13.1 22 PF3D7_1115400 cysteine proteinase falcipain 3 (FP3) 3 13 4 PF3D7_0708500 heat shock protein 86 family protein 19 12.8 31 deoxyribodipyrimidine photo-lyase, PF3D7_0513600 putative 8 12.8 10 PF3D7_1025000 formin 2, putative 14 12.7 17 mannose-6-phosphate isomerase, PF3D7_0801800 putative 9 12.7 12 serine/threonine protein kinase, FIKK PF3D7_0424500 family (FIKK4.1) 5 12.7 6 H/ACA ribonucleoprotein complex PF3D7_1417500 subunit 4, putative (CBF5) 4 12.7 5 conserved Plasmodium protein, unknown PF3D7_1024000 functio 4 12.7 4 DNA replication licensing factor MCM3, PF3D7_0527000 putative (MCM3) 9 12.6 11 DNA replication licensing factor MCM4 PF3D7_1317100 (MCM4) 6 12.5 7 polypyrimidine tract binding protein, PF3D7_0606500 putative 5 12.5 6 conserved Plasmodium protein, unknown PF3D7_1366900 function 5 12.4 4 PF3D7_1107700 pescadillo-like protein (PES) 5 12.4 6 pre-mRNA-processing protein 45, PF3D7_0218700 putative (PRP45) 3 12.4 3 conserved Plasmodium protein, unknown PF3D7_1127800 function 14 12.3 16 PF3D7_1353400 Ran-binding protein, putative 9 12.3 10 conserved Plasmodium protein, unknown PF3D7_1314700 function 6 12.1 6 rRNA-processing protein EBP2, putative PF3D7_1028300 (EBP2) 3 12.1 3 glutamate dehydrogenase, putative PF3D7_0802000 (GDH3 8 12 7 Plasmodium exported protein (PHISTb), PF3D7_0401800 unknown function (PfD80) 7 12 7

choline/ethanolaminephosphotransferase PF3D7_0628300 , putative (CEPT) 3 11.8 3 regulator of chromosome condensation, PF3D7_0711500 putative 7 11.6 9 conserved Plasmodium protein, unknown PF3D7_0923400 function 15 11.5 20 PF3D7_0622200 radical SAM protein, putative 6 11.5 6 PF3D7_0731600;PF 3D7_0215300 acyl-CoA synthetase (ACS5) 5 11.5 5 PF3D7_1104100 syntaxin, Qa-SNARE family (SYN13) 3 11.5 2 141

ribose-phosphate pyrophosphokinase, PF3D7_1327800 putative 4 11.4 5 PF3D7_0525100 acyl-CoA synthetase (ACS10) 4 11.4 5 Plasmodium exported protein, unknown PF3D7_1401200 function 3 11.4 4 large subunit rRNA methyltransferase, PF3D7_1354300 putative 10 11.2 11 PF3D7_0622800 leucine--tRNA ligase, putative 9 11.2 8 PF3D7_0214000 T-complex protein 1, putative 4 11.1 4 high molecular weight rhoptry protein 3 PF3D7_0905400 (RhopH3) 6 10.9 6 PF3D7_0708800 heat shock protein 110 (HSP110c) 6 10.9 8 conserved Plasmodium protein, unknown PF3D7_0724100 function 12 10.8 15 PF3D7_1220100 pre-mRNA splicing factor, putative 5 10.7 6 PF3D7_1105700 conserved protein, unknown function 4 10.7 4 cAMP-dependent protein kinase PF3D7_1223100 regulatory subunit (PKAr) 3 10.7 4 PF3D7_1149200 ring-infected erythrocyte surface antigen 13 10.6 7 conserved Plasmodium protein, unknown PF3D7_1450700 function 5 10.6 6 PF3D7_1226700 conserved protein, unknown function 5 10.5 6 nuclear protein localization protein 4, PF3D7_0507700 putative (NPL4) 4 10.5 5 pre-mRNA-processing-splicing factor 8, PF3D7_0405400 putative (PRPF8) 20 10.4 22 conserved Plasmodium protein, unknown PF3D7_1138700 function 20 10.4 26 PF3D7_0907600 translation initiation factor SUI1, putative 5 10.3 4 PF3D7_1470600 RAP protein, putative 4 10.3 5 eukaryotic translation initiation factor 5, PF3D7_1206700 putative 4 10.1 4 apurinic/apyrimidinic endonuclease Apn1, PF3D7_1332600 putative (APN1) 3 9.9 3 protein arginine N-methyltransferase 5, PF3D7_1361000 putative (PRMT5) 5 9.7 5 PF3D7_1311900 vacuolar ATP synthase subunit a (vapA) 3 9.7 4 PF3D7_0417300 LETM1-like protein, putative 5 9.6 6 conserved Plasmodium protein, unknown PF3D7_0912400 function 3 9.6 2 structural maintenance of chromosome PF3D7_1130700 protein, putative 11 9.5 11 splicing factor 3A subunit 3, putative PF3D7_0924700 (SF3A3) 5 9.5 5 gamma-glutamylcysteine synthetase PF3D7_0918900 (gammaGCS) 7 9.4 8 PF3D7_1308200 carbamoyl phosphate synthetase (cpsSII) 10 9.3 11 golgi organization and biogenesis factor, PF3D7_0418200 putative 3 9.3 3 142

PF3D7_0720400 ferrodoxin reductase-like protein 3 9.3 3 conserved Plasmodium protein, unknown PF3D7_0111800 function 5 9.2 6 PF3D7_0810800 dihydropteroate synthetase (DHPS) 4 9.2 5 eukaryotic translation initiation factor 2 PF3D7_1410600 gamma subunit, putative 3 9.2 3 PF3D7_0613700 syntaxin binding protein, putative 3 9.1 3 PF3D7_0524000 karyopherin beta (KASbeta) 6 8.9 6 PF3D7_0727200 cysteine desulfurase, putative (NFS) 3 8.9 3 vacuolar transporter chaperone, putative, PF3D7_1230200 pseudogene 8 8.8 9 conserved Plasmodium protein, unknown PF3D7_1133700 function 7 8.6 7 PF3D7_1216900 DNA-binding chaperone, putative 7 8.6 10 PF3D7_0625300 DNA polymerase 1, putative 7 8.6 8 N-alpha-acetyltransferase 15, NatA PF3D7_1244100 auxiliary subunit, putative 7 8.6 6 CCR4-NOT transcription complex subunit PF3D7_1235300 4, putative (NOT4) 9 8.5 10 PF3D7_1105600 translocon component PTEX88 (PTEX88) 4 8.5 3 PF3D7_0623100 coronin binding protein, putative 3 8.5 4 Plasmodium exported protein, unknown PF3D7_0113200 function 3 8.3 3 PF3D7_1108000 IWS1-like protein, putative 3 8.3 3 PF3D7_1219100 clathrin heavy chain, putative 10 8.2 12 conserved Plasmodium protein, unknown PF3D7_0621700 function 7 8 6 conserved Plasmodium protein, unknown PF3D7_1422400 function 7 8 7 U3 snoRNA-associated small subunit rRNA processing associated protein, PF3D7_1333600 putative 5 7.9 5 NLI interacting factor-like phosphatase, PF3D7_1012700 putative (NIF4) 8 7.8 8 conserved Plasmodium protein, unknown PF3D7_0821000 function 3 7.8 3 PF3D7_1445600 RNA-binding protein, putative 4 7.7 6 PF3D7_1405900 RNA-binding protein, putative 5 7.6 7 FACT complex subunit SPT16, putative PF3D7_0517400 (FACT-L) 5 7.6 6 pre-mRNA-processing ATP-dependent PF3D7_0508700 RNA helicase PRP5, putative (PRP5) 12 7.4 19 PF3D7_0903400 DEAD/DEAH box helicase, putative 12 7.4 13 U4/U6.U5 tri-snRNP-associated protein 1, PF3D7_0323700 putative (SART1) 4 7.4 4 PF3D7_0515000 0RNA-binding protein, putative 4 7.4 5 beta subunit of coatomer complex, PF3D7_0905900 putative (SEC27) 4 7.4 3 PF3D7_1124300 conserved Plasmodium protein, unknown 3 7.4 3 143

function PF3D7_1110900 ES2 protein, putative 3 7.3 3 PF3D7_0823300 histone acetyltransferase GCN5 (GCN5) 8 7.2 8 peptidyl-prolyl cis-trans isomerase PF3D7_0510200 (CYP87) 3 7.2 4 PF3D7_0308200 TCP-1/cpn60 chaperonin family, putative 3 7.2 3 conserved Plasmodium protein, unknown PF3D7_1134200 function 10 7.1 11 PF3D7_0612900 nucleolar GTP-binding protein 1, putative 4 7 6 PF3D7_1340300 nucleolar complex protein 2, putative 4 7 5 PF3D7_1022600 kelch protein, putative 4 7 4 conserved Plasmodium protein, unknown PF3D7_1225600 function 5 6.9 7 transcription factor with AP2 domain(s), PF3D7_1449500 putative (ApiAP2) 3 6.9 3 conserved Plasmodium protein, unknown PF3D7_1468100 function 10 6.8 11 PF3D7_0906600 zinc finger protein, putative 5 6.8 5 conserved Plasmodium protein, unknown PF3D7_1237200 function 5 6.8 3 DNA-directed RNA polymerase II second PF3D7_0215700 largest subunit, putative 5 6.6 6 PF3D7_0302500;PF cytoadherence linked asexual protein 3.1 3D7_0302200 (CLAG3.1) 5 6.6 6 PF3D7_1145400 dynamin-like protein (DYN1) 4 6.6 5 heat shock protein 101,chaperone PF3D7_1116800 protein ClpB2 (HSP101) 3 6.6 3 N2,N2-dimethylguanosine tRNA PF3D7_1319300 methyltransferase, putative 3 6.6 3 multidrug resistance protein 2 (heavy PF3D7_1447900 metal transport family) (MDR2) 5 6.4 5 conserved Plasmodium protein, unknown PF3D7_0723400 function 7 6.3 8 PF3D7_0523000 multidrug resistance protein (MDR1) 6 6.3 6 U3 small nucleolar ribonucleoprotein PF3D7_1027100 protein MPP10, putative (MPP10) 3 6.3 3 PF3D7_1334200 chaperone binding protein, putative 3 6.3 3 PF3D7_1138500 protein phosphatase 2C (PP2C) 3 6.3 3 conserved Plasmodium protein, unknown PF3D7_0609700 function 12 6.2 14 non-SERCA-type Ca2+ -transporting P- PF3D7_1211900 ATPase (ATP4) 4 6.2 5 40S ribosomal processing protein, PF3D7_0409200 putative 3 6 3 histone S-adenosyl methyltransferase, PF3D7_1227800 putative 5 5.9 6 conserved Plasmodium protein, unknown PF3D7_1126500 function 5 5.8 6 PF3D7_1233900 sentrin-specific protease 1 (SENP1) 4 5.8 5 144

DNA replication licensing factor MCM5, PF3D7_1211700 putative (MCM5) 3 5.8 3 splicing factor 3A subunit 1, putative PF3D7_1474500 (SF3A1) 3 5.8 3 conserved Plasmodium protein, unknown PF3D7_0604500 function 13 5.7 16 DNA-directed RNA polymerase II subunit PF3D7_0318200 RPB1, putative (RPB1) 8 5.7 7 conserved Plasmodium protein, unknown PF3D7_1234100 function 12 5.4 17 PF3D7_1332900 isoleucine--tRNA ligase, putative 5 5.4 6 PF3D7_0528100 AP-1 complex subunit beta, putative 3 5.4 4 PF3D7_0930300 merozoite surface protein 1 (MSP1) 7 5.3 6 PF3D7_0727800 cation transporting ATPase, putative 6 5.3 6 splicing factor 3B subunit 3, putative PF3D7_1234800 (SF3B3) 4 5.3 5 conserved Plasmodium protein, unknown PF3D7_0807600 function 8 5.2 11 transcription factor with AP2 domain(s) PF3D7_1007700 (ApiAP2) 6 5.2 8 PF3D7_1118300 1118300 | insulinase, putative 5 5.2 4 PF3D7_1238300 cell cycle control protein, putative 4 5.1 4 pre-mRNA-processing factor 40, putative PF3D7_1316500 (PRP40) 3 5 3 conserved Plasmodium protein, unknown PF3D7_1457300 function 3 5 3

3D7attB Sample 2 Sequence MS/ coverage MS Protein IDs Fasta headers Peptides [%] count glyceraldehyde-3-phosphate PF3D7_1462800 dehydrogenase (GAPDH) 28 86.1 40 PF3D7_1357100;PF3D7 _1357000 elongation factor 1-alpha 38 76.5 76 PF3D7_1006800 RNA-binding protein, putative 24 75.2 46 PF3D7_0929200 RNA-binding protein, putative 25 73 26 PF3D7_1144000 40S ribosomal protein S21 (RPS21) 6 70.7 10 PF3D7_0826700 receptor for activated c kinase (RACK) 14 70.6 27 eukaryotic translation initiation factor 3 PF3D7_1007900 subunit 7, putative 46 69.2 50 PF3D7_1026800 40S ribosomal protein S2 (RPS2) 14 68.4 18 PF3D7_0408300 zinc finger, RAN binding protein, putative 23 68.3 35 conserved Plasmodium protein, unknown PF3D7_1230800 function 52 68.2 68 PF3D7_1325100 phosphoribosylpyrophosphate synthetase 23 67.5 31 PF3D7_1019400 60S ribosomal protein L30e, putative 6 66.7 10 PF3D7_0422400 40S ribosomal protein S19 (RPS19) 14 65.9 22 PF3D7_1108700 heat shock protein DnaJ homologue Pfj2 37 65.6 56 145

PF3D7_0502100 HCNGP-like protein 12 65.4 9 PF3D7_1426000 60S ribosomal protein L21 (RPL21) 13 65.2 21 PF3D7_0626800 pyruvate kinase (PyrK) 24 65 29 PF3D7_0322900 40S ribosomal protein S3A, putative 20 64.9 38 PF3D7_1421200 40S ribosomal protein S25 (RPS25) 11 64.8 14 0Plasmodium exported protein (PHISTb), PF3D7_0731300 unknown function (PfG174) 22 64 53 PF3D7_1142500 60S ribosomal protein L28 (RPL28) 11 62.2 15 PF3D7_0922200 S-adenosylmethionine synthetase (SAMS) 19 61.9 24 PF3D7_0818200 14-3-3 protein (14-3-3I) 8 61.1 11 PF3D7_1407100 fibrillarin, putative (NOP1) 17 60.4 19 PF3D7_0721600 40S ribosomal protein S5, putative 9 60 16 PF3D7_0813900 40S ribosomal protein S16, putative 9 59.7 9 PF3D7_0818900;PF3D7 _0831700 heat shock protein 70 (HSP70) 49 58.9 105 PF3D7_0516200 40S ribosomal protein S11 5 58.9 11 PF3D7_0719700 40S ribosomal protein S10, putative 8 58.4 20 pre-mRNA-splicing factor ATP-dependent PF3D7_1030100 RNA helicase PRP22, putative (PRP22) 73 58.3 52 vesicle-associated membrane protein, PF3D7_1439800 putative 9 58.1 9 PF3D7_0617800 histone H2A (H2A) 7 57.6 11 PF3D7_0316800 40S ribosomal protein S15A, putative 8 56.9 12 PF3D7_1424100 60S ribosomal protein L5, putative 19 56.8 50 PF3D7_0415500 nuclear cap-binding protein, putative 14 56.8 19 PF3D7_1002400.1;PF3D 7_1002400.2 RNA-binding protein, putative 24 55.2 38 PF3D7_0527900 RNA helicase 1 40 54.3 50 PF3D7_0507100 60S ribosomal protein L4 (RPL4) 21 54.3 37 PF3D7_0917900 heat shock protein 70 (HSP70-2) 29 54.1 36 PF3D7_1105400 40S ribosomal protein S4, putative 12 54 26 PF3D7_1011800 PRE-binding protein (PREBP) 73 53.9 162 PF3D7_0610400;PF3D7 _0617900 histone H3 (H3) 6 53.7 5 PF3D7_1134100 protein disulfide isomerase (PDI-11) 22 53 43 PF3D7_1414300 60S ribosomal protein L10, putative 13 53 24 PF3D7_0823200 RNA-binding protein, putative 14 52.8 23 PF3D7_0812500 RNA-binding protein, putative 71 52.5 61 PF3D7_0516900 60S ribosomal protein L2 (RPL2) 12 51.9 28 PF3D7_1358800 40S ribosomal protein S15 (RPS15) 11 51.7 19 PF3D7_1447000 40S ribosomal protein S5 13 51.5 25 PF3D7_0517300 pre-mRNA-splicing factor (SR1) 23 51.3 42 PF3D7_1126200 40S ribosomal protein S18, putative 7 51.3 13 PF3D7_0608800 ornithine aminotransferase (OAT) 16 51 31 PF3D7_1460700 60S ribosomal protein L27 (RPL27) 10 50.7 21 PF3D7_1472000 pre-mRNA-splicing factor ISY1, putative 6 50.7 10 146

conserved Plasmodium protein, unknown PF3D7_1445700 function 15 50.4 13 PF3D7_1136300 tudor staphylococcal nuclease (TSN) 45 49.8 51 PF3D7_1238100 1 calcyclin binding protein, putative 11 48.7 10 PF3D7_1341200 60S ribosomal protein L18, putative 11 48.4 22 conserved Plasmodium protein, unknown PF3D7_0617200 function 28 48 28 PF3D7_1302800 40S ribosomal protein S7, putative 6 47.9 13 40S ribosomal protein S11, putative PF3D7_0317600 (RPS11) 9 47.8 18 PF3D7_1004400 RNA-binding protein, putative 74 47.7 92 eukaryotic translation initiation factor, PF3D7_0815600 putative 9 47.4 4 conserved Plasmodium protein, unknown PF3D7_1305900 function 23 46.7 23 PF3D7_1307800 fork head domain protein, putative 31 46.3 17 1367100 | U1 small nuclear PF3D7_1367100 ribonucleoprotein, putative 23 46.3 21 PF3D7_0517000 60S ribosomal protein L12, putative 8 46.1 9 PF3D7_1408600 40S ribosomal protein S8e, putative 10 45.4 15 PF3D7_0618300 60S ribosomal protein L27a, putative 5 45.3 7 multiprotein bridging factor type 1, putative PF3D7_1128200 (MBF1 7 44.9 8 PF3D7_0814200 DNA/RNA-binding protein Alba 1 (ALBA1) 12 44.8 18 PF3D7_1360900 polyadenylate-binding protein, putative 14 44.7 24 eukaryotic translation initiation factor 5A PF3D7_1204300 (EIF5A) 9 44.7 11 PF3D7_0915400 6-phosphofructokinase (PFK9) 49 44.3 101 pre-mRNA-splicing factor ATP-dependent PF3D7_1231600 RNA helicase PRP2, putative (PRP2) 38 44.3 38 PF3D7_1104400 conserved protein, unknown function 15 44.1 31 PF3D7_1008700 tubulin beta chain 11 44 12 PF3D7_0217800 40S ribosomal protein S26 (RPS26) 4 43.9 6 PF3D7_0319500 RNA-binding protein, putative 11 43.7 11 PF3D7_0814000 60S ribosomal protein L13-2, putative 8 43.7 13 PF3D7_1136800 DnaJ protein, putative 14 43.4 6 PF3D7_1437900 HSP40, subfamily A, putative 10 43.4 29 PF3D7_0312800 60S ribosomal protein L26, putative 7 42.9 10 eukaryotic translation initiation factor 3 PF3D7_1212700 subunit 10, putative 65 42.3 67 AAA family ATPase, CDC48 subfamily PF3D7_0711000 (Cdc48) 47 42.1 105 PF3D7_1105100 histone H2B (H2B) 9 41.9 18 PF3D7_1027800 60S ribosomal protein L3 (RPL3) 16 41.5 33 PF3D7_0903900 60S ribosomal protein L32 (RPL32) 7 40.5 13 PF3D7_1338200 60S ribosomal protein L6-2, putative 6 40.3 12 PF3D7_1331800 60S ribosomal protein L23, putative 3 40.3 12 147

PF3D7_1015900 enolase (ENO) 12 39.9 22 PF3D7_0730900 EMP1-trafficking protein (PTP4) 20 39.5 39 PF3D7_1465900 40S ribosomal protein S3 9 38.9 16 PF3D7_1451100 elongation factor 2 23 38.2 40 PF3D7_1120100 phosphoglycerate mutase, putative (PGM1) 7 38 8 PF3D7_0417800 cdc2-related protein kinase 1 (CRK1) 28 37.8 33 PF3D7_1109900 60S ribosomal protein L36 (RPL36) 6 37.5 13 PF3D7_1468700 eukaryotic initiation factor 4A (eIF4A) 10 36.9 23 Plasmodium exported protein (PHISTb), PF3D7_0424600 unknown function 8 36.9 12 PF3D7_1134000 heat shock protein 70 (HSP70-3) 19 36.7 31 PF3D7_1008800 nucleolar protein 5, putative (NOP5) 11 36.5 12 PF3D7_0708400 heat shock protein 90 (HSP90) 17 36.1 89 U4/U6 snRNA-associated-splicing factor, PF3D7_0920900 putative (PRP24) 14 36 15 ATP-dependent RNA helicase DDX5, PF3D7_1445900 putative (DDX5 14 35.7 23 PF3D7_1342000 40S ribosomal protein S6 12 35.6 17 PF3D7_0922500 phosphoglycerate kinase (PGK) 9 35.3 15 conserved Plasmodium protein (10b PF3D7_1021900 antigen), unknown function 76 35.2 44 PF3D7_1242700 40S ribosomal protein S17, putative 4 35 7 PF3D7_1243600 translation initiation factor SUI1, putative 4 34.8 5 PF3D7_1003500 40S ribosomal protein S20e, putative 4 34.7 7 pre-mRNA-splicing factor 38A, putative PF3D7_1132600 (PRP38A) 15 34.5 5 PF3D7_1471100 exported protein 2 (EXP2) 9 34.5 15 PF3D7_0817900 high mobility group protein B2 (HMGB2) 3 34.3 7 0801000 | Plasmodium exported protein (PHISTc), unknown function | 08_v3:84568- PF3D7_0801000 88422(-) | length=1219 | 24 34 39 conserved Plasmodium protein, unknown PF3D7_1419700 function| 13 34 20 PF3D7_0629200 DnaJ protein, putative 9 33.9 10 PF3D7_1431700 60S ribosomal protein L14, putative 5 33.3 7 conserved Plasmodium protein, unknown PF3D7_1423700 function 56 33.1 35 PF3D7_1220900 heterochromatin protein 1 (HP1) 5 33.1 5 S-adenosyl-L-homocysteine hydrolase PF3D7_0520900 (SAHH) 14 33 10 PF3D7_0320900 histone H2A variant, putative (H2A.Z) 3 32.9 4 splicing factor U2AF small subunit, putative PF3D7_1119300 (U2AF1) 6 32.7 15 PF3D7_0304400 60S ribosomal protein L44 (RPL44) 5 32.7 9 PF3D7_0923900 RNA-binding protein, putative 4 32.7 4 conserved Plasmodium protein, unknown PF3D7_1036900 function 65 32.5 44 148

hypoxanthine-guanine PF3D7_1012400 phosphoribosyltransferase (HGPRT) 3 32 3 PF3D7_0307200 60S ribosomal protein L7, putative 6 31.9 18 debranching enzyme-associated PF3D7_1220400 ribonuclease, putative (DRN1) 17 31.8 17 ATP-dependent RNA helicase UAP56 PF3D7_0209800 (UAP56) 7 31.5 8 PF3D7_1323400 60S ribosomal protein L23 (RPL23) 7 31.1 18 PF3D7_1136500.1;PF3D 7_1136500.2 casein kinase 1 (CK1) 6 31 11 eukaryotic translation initiation factor 3 PF3D7_0918300 subunit 5, putative 5 30.9 4 PF3D7_1424400 60S ribosomal protein L7-3, putative 9 30.7 17 PF3D7_1351400 60S ribosomal protein L17, putative 6 30.5 10 phosphoethanolamine N- PF3D7_1343000 methyltransferase (PMT) 6 30.5 5 conserved Plasmodium protein, unknown PF3D7_0924100 function 22 30.2 32 PF3D7_1236100 clustered-asparagine-rich protein 7 30.1 8 PF3D7_1015600 heat shock protein 60 (HSP60) 15 30 15 PF3D7_1341300 60S ribosomal protein L18-2, putative 3 29.9 9 PF3D7_0107000 centrin-1 (CEN1) 3 29.8 2 PF3D7_0409400 heat shock protein 40 (DnaJ) 14 29.5 32 PF3D7_1246200 actin I (ACT1) 8 29.5 12 PF3D7_1338300 elongation factor 1-gamma, putative 6 29.4 26 serine/arginine-rich splicing factor 4 PF3D7_1022400 (SRSF4) 22 28.8 52 PF3D7_1108400 casein kinase 2, alpha subunit (CK2alpha) 6 28.7 16 PF3D7_0503400 actin-depolymerizing factor 1 (ADF1) 3 28.7 6 conserved Plasmodium protein, unknown PF3D7_1120000 function 16 28.6 21 PF3D7_1117700 GTP-binding nuclear protein RAN/TC4 (RAN) 4 28.5 12 conserved Plasmodium protein, unknown PF3D7_1459400 function 5 28.2 9 PF3D7_0210100.1 60S ribosomal protein L37ae, putative 3 28.1 3 PF3D7_1104100 syntaxin, Qa-SNARE family (SYN13) 6 28 2 conserved Plasmodium protein, unknown PF3D7_1350300 function 5 27.9 1 PF3D7_0303200 HAD superfamily protein, putative 25 27.6 36 PF3D7_1345100 1 thioredoxin 2 (TRX2) 6 27.4 7 Plasmodium exported protein (PHISTb), PF3D7_0532300 unknown function 8 27.3 0 sin3 associated polypeptide p18-like PF3D7_0711400 protein 31 27.1 27 Plasmodium exported protein, unknown PF3D7_1149400 function 5 26.8 6 PF3D7_0503800 60S ribosomal protein L31 (RPL31) 3 26.7 6 PF3D7_0823800 DnaJ protein, putative 12 26.3 20 149

0921900 | conserved Plasmodium protein, PF3D7_0921900 unknown function 4 26.2 5 eukaryotic translation initiation factor 3 PF3D7_1206200 subunit 8, putative 23 26 26 Plasmodium exported protein (PHISTc), PF3D7_1148700 unknown function (GEXP12) 6 25.9 16 1315400 | zinc finger (CCCH type) protein, PF3D7_1315400 putative 8 25.8 14 PF3D7_0605100 RNA-binding protein, putative 16 25.7 22 serine/arginine-rich splicing factor 12 PF3D7_0503300 (SRSF12) 16 25.6 25 PF3D7_1317800 40S ribosomal protein S19 (RPS19) 4 25.5 12 knob-associated histidine-rich protein PF3D7_0202000 (KAHRP) 17 25.4 51 PF3D7_0903700;PF3D7 _0422300 alpha tubulin 1 7 25.2 8 GrpE protein homolog, mitochondrial, PF3D7_1124700 putative (MGE1) 5 25.2 6 PF3D7_1005500 regulator of nonsense transcripts, putative 23 25.1 18 conserved Plasmodium protein, unknown PF3D7_1366300 function 77 25 13 conserved Plasmodium protein, unknown PF3D7_0722000 function 8 24.9 5 PF3D7_1222300 endoplasmin, putative (GRP94) 14 24.7 21 PF3D7_1326300 RNA-binding protein, putative 15 24.3 36 ADP/ATP transporter on adenylate PF3D7_1037300 translocase (ADT) 6 24.3 9 PF3D7_1415300 RNA-binding protein Nova-1, putative 5 24.3 4 eukaryotic translation initiation factor 2 PF3D7_1010600 beta subunit, putative 3 23.9 7 PF3D7_0706400 60S ribosomal protein L37 (RPL37) 3 23.9 6 conserved Plasmodium protein, unknown PF3D7_0706500 function 35 23.8 22 PF3D7_0708500 heat shock protein 86 family protein 27 23.8 31 PF3D7_1352500 thioredoxin-related protein, putative 4 23.6 7 PF3D7_0211800 asparagine--tRNA ligase (AsnRS) 10 23.3 12 PF3D7_0213100;PF3D7 _0501100.2;PF3D7_050 1100.1 heat shock protein 40, putative 7 23.2 8 regulator of chromosome condensation, PF3D7_0711500 putative 13 23.1 9 conserved Plasmodium protein, unknown PF3D7_1244700 function 5 23 3 PF3D7_1233900 sentrin-specific protease 1 (SENP1) 21 22.7 5 PF3D7_1452700 U1 snRNA associated protein, putative 6 22.6 18 PF3D7_0812400 karyopherin alpha (KARalpha) 7 22.4 11 nuclear preribosomal assembly protein, PF3D7_1124800 putative 4 22.4 10 PF3D7_0218700 0 pre-mRNA-processing protein 45, putative 6 21.8 3 150

(PRP45) conserved Plasmodium protein, unknown PF3D7_0813000 function 16 21.7 20 conserved Plasmodium protein, unknown PF3D7_0801500 function 5 21.7 8 polyadenylate-binding protein, putative PF3D7_1224300 (PABP) 11 21.5 10 conserved Plasmodium protein, unknown PF3D7_1330800 function 7 21.5 22 conserved Plasmodium protein, unknown PF3D7_1415400 function 29 21.4 7 small subunit rRNA processing factor, PF3D7_1358100 putative 20 21.1 20 small subunit rRNA processing factor, PF3D7_1207100 putative 20 21 30 1 vacuolar transporter chaperone, putative, PF3D7_1230200 pseudogene 17 21 9 Plasmodium exported protein (PHISTa), PF3D7_0402000 unknown function 13 21 37 PF3D7_0609200 citrate synthase-like protein, putative 8 20.9 9 PF3D7_0827900 protein disulfide isomerase (PDI8) 5 20.9 6 PF3D7_1110400 asparagine-rich antigen 37 20.7 49 conserved Plasmodium protein, unknown PF3D7_0403200 function 18 20.7 17 eukaryotic translation initiation factor 3, PF3D7_0528200 subunit 6, putative 7 20.7 4 Plasmodium exported protein (PHISTc), PF3D7_1001700 unknown function 3 20.7 3 splicing factor 1 (SF1) | 13_v3:902383- PF3D7_1321700 905166(+) 17 20.6 38 PF3D7_1311800 M1-family alanyl aminopeptidase (M1AAP) 12 20.6 11 lysine-rich membrane-associated PHISTb PF3D7_0532400 protein (LyMP) 8 20.6 0 PF3D7_1202900 high mobility group protein B1 (HMGB1) 3 20.6 9 conserved Plasmodium protein, unknown PF3D7_1406200 function 45 20.4 75 PF3D7_1309100 60S ribosomal protein L24, putative 5 20.4 7 ATP-dependent zinc metalloprotease FTSH PF3D7_1239700 1 (FTSH1) 12 20.3 20 PF3D7_1224000 GTP cyclohydrolase I (GCH1) 5 20.1 6 conserved Plasmodium protein, unknown PF3D7_0813300 function 7 20 17 conserved Plasmodium protein, unknown PF3D7_0724100 function 29 19.9 15 isocitrate dehydrogenase (NADP), PF3D7_1345700 mitochondrial precursor (IDH) 6 19.9 7 regulator of nonsense transcripts 3B, PF3D7_1327700 putative (UPF3B) 3 19.9 1 1conserved Plasmodium protein, unknown PF3D7_1026000 function 6 19.8 7 151

PF3D7_0614500 | 60S ribosomal protein L19 (RPL19) 3 19.8 7 conserved Plasmodium protein, unknown PF3D7_0508600 function 3 19.8 2 PF3D7_1349200 glutamate--tRNA ligase, putative 10 19.7 12 0722600 | nucleolar rRNA processing PF3D7_0722600 protein, putative 8 19.6 14 PF3D7_1246600 pre-mRNA-splicing factor, putative 7 19.5 6 PF3D7_1474900 trailer hitch homolog, putative (CITH) 5 19.5 2 0923400 | conserved Plasmodium protein, PF3D7_0923400 unknown function 24 19.3 20 PF3D7_1324900 L-lactate dehydrogenase (LDH) 4 19.3 4 PF3D7_1445400 protein serine/threonine kinase-1 (CLK1) 17 19 13 conserved Plasmodium protein, unknown PF3D7_1450700 function 8 19 6 PF3D7_0415900 60S ribosomal protein L15, putative 4 19 7 PF3D7_1334500 MSP7-like protein (MSRP6) 12 18.9 17 high molecular weight rhoptry protein 2 PF3D7_0929400 (RhopH2) 12 18.9 14 PF3D7_1004000 60S ribosomal protein L13, putative 6 18.8 7 Plasmodium exported protein, unknown PF3D7_1401200 function 5 18.6 4 PF3D7_0310300 phosphoglycerate mutase, putative 14 18.5 15 PF3D7_0406100 vacuolar ATP synthase subunit b 5 18.4 9 1133700 | conserved Plasmodium protein, PF3D7_1133700 unknown function 12 18.2 7 PF3D7_1472900 dihydroorotase, putative 4 18.2 7 PF3D7_1141800 phd finger protein, putative 5 18 0 transcription elongation factor SPT5, PF3D7_0610900 putative (SPT5) 30 17.9 29 conserved Plasmodium protein, unknown PF3D7_1314700 function 9 17.9 6 RNA-binding protein musashi, putative PF3D7_0916700 (HoMu) 6 17.8 11 PF3D7_1311400 AP-1 complex subunit mu, putative 5 17.8 8 signal recognition particle receptor, beta PF3D7_1246800 subunit (SRPR-beta) 3 17.6 3 pre-mRNA-splicing factor 38B, putative PF3D7_1407300 (PRP38B) 8 17.5 7 ribose-phosphate pyrophosphokinase, PF3D7_1327800 putative 5 17.5 5 conserved Plasmodium protein, unknown PF3D7_1409600 function 7 17.4 8 Plasmodium exported protein (PHISTb), PF3D7_1476200 unknown function 5 17.4 14 PF3D7_1353400 Ran-binding protein, putative 17 17.3 10 conserved Plasmodium protein, unknown PF3D7_1105800 function 4 17.3 6 conserved Plasmodium protein, unknown PF3D7_1025300 function 14 17.2 23 152

PF3D7_1453700 co-chaperone p23 (P23) 3 17.1 5 nuclear protein localization protein 4, PF3D7_0507700 putative (NPL4) 5 16.9 5 PF3D7_0520000 40S ribosomal protein S9, putative 3 16.9 8 conserved Plasmodium protein, unknown PF3D7_0723900 function 19 16.8 24 PF3D7_1034900 methionine--tRNA ligase, putative 10 16.8 15 conserved Plasmodium protein, unknown PF3D7_0521000 function 10 16.8 12 PF3D7_0922100 ubiquitin-like protein, putative 18 16.3 26 PF3D7_0311100 pre-mRNA splicing factor, putative 13 16.3 15 conserved Plasmodium protein, unknown PF3D7_1366900 function 9 16.2 4 PF3D7_0315800 zinc finger protein, putative 3 16.2 1 PF3D7_1119000 acyl-CoA-binding protein, putative | 3 16.2 2 conserved Plasmodium protein, unknown PF3D7_0604500 function 37 16 16 conserved Plasmodium protein, unknown PF3D7_0307700 function 23 15.9 7 Plasmodium exported protein (PHISTb), PF3D7_0401800 unknown function (PfD80) 9 15.7 7 conserved Plasmodium protein, unknown PF3D7_1225600 function 10 15.6 7 deoxyribodipyrimidine photo-lyase, PF3D7_0513600 putative 11 15.4 10 PF3D7_1227100 DNA helicase 60 (DH60) 7 15.4 12 PF3D7_0818000 conserved protein, unknown function 3 15.3 3 serine/threonine protein kinase, FIKK PF3D7_0805700 family (FIKK8) 14 15 5 PF3D7_1434600 methionine aminopeptidase 2 (METAP2) 6 15 11 conserved Plasmodium protein, unknown PF3D7_0723400 function 20 14.8 8 golgi organization and biogenesis factor, PF3D7_0418200 putative 5 14.7 3 eukaryotic translation initiation factor 3 PF3D7_0716800 37.28 kDa subunit, putative 3 14.7 3 PF3D7_0810600 RNA helicase, putative 8 14.6 13 polyadenylate-binding protein-interacting PF3D7_1107300 protein 1, putative (PAIP1) 44 14.5 15 conserved Plasmodium protein, unknown PF3D7_1147300 function 11 14.5 15 eukaryotic translation initiation factor PF3D7_1438000 eIF2A, putative 5 14.4 19 polypyrimidine tract binding protein, PF3D7_0606500 putative 5 13.9 6 PF3D7_1130200 60S ribosomal protein P0 (PfP0) 3 13.9 11 autophagy-related protein 18, putative PF3D7_1012900 (ATG18) 4 13.7 5 conserved Plasmodium protein, unknown PF3D7_0210900 function 3 13.7 2 153

PF3D7_1357800 TCP-1/cpn60 chaperonin family, putative 3 13.6 22 pre-mRNA-processing-splicing factor 8, PF3D7_0405400 putative (PRPF8) 27 13.2 22 ring-infected erythrocyte surface antigen PF3D7_0102200 (RESA) 16 13 24 PF3D7_0722400 GTP-binding protein, putative 3 13 8 phosphoacetylglucosamine mutase, PF3D7_1130000 putative (PAGM) 8 12.9 13 cAMP-dependent protein kinase regulatory PF3D7_1223100 subunit (PKAr) 3 12.9 4 PF3D7_0624000 hexokinase (HK) 4 12.8 18 PF3D7_1212900 bromodomain protein, putative 11 12.6 16 PF3D7_1444800 fructose-bisphosphate aldolase (FBPA) 3 12.2 14 high molecular weight rhoptry protein 3 PF3D7_0905400 (RhopH3) 6 12 6 splicing factor U2AF large subunit, putative PF3D7_1468800 (U2AF2 5 11.8 0 conserved Plasmodium protein, unknown PF3D7_0729100 function 19 11.7 3 conserved Plasmodium protein, unknown PF3D7_1031300 function ] 7 11.4 1 conserved Plasmodium protein, unknown PF3D7_0807600 function 14 11.2 11 PF3D7_0813600 translation initiation factor SUI1, putative 5 11.2 17 PF3D7_1445600 RNA-binding protein, putative 5 11.1 6 conserved Plasmodium protein, unknown PF3D7_0214400 function 4 11.1 6 DEAD/DEAH box ATP-dependent RNA PF3D7_1251500 helicase, putative 5 10.9 10 PF3D7_1409800 RNA binding protein Bruno, putative (HoBo) 4 10.9 9 eukaryotic translation initiation factor, PF3D7_0517700 putative 4 10.8 16 PF3D7_0414000 chromosome associated protein, putative 8 10.6 11 conserved Plasmodium membrane protein, PF3D7_0505700 unknown function 4 10.6 1 conserved Plasmodium protein, unknown PF3D7_1225400 function 3 10.6 1 DNA replication licensing factor MCM3, PF3D7_0527000 putative (MCM3) 5 9.8 11 PF3D7_0607000 translation initiation factor IF-2, putative 7 9.7 13 PF3D7_1220100 pre-mRNA splicing factor, putative 6 9.7 6 U4/U6.U5 tri-snRNP-associated protein 1, PF3D7_0323700 putative (SART1) 6 9.7 4 PF3D7_0907600 translation initiation factor SUI1, putative 5 9.7 4 DNA replication licensing factor MCM4 PF3D7_1317100 (MCM4) 4 9.7 7 PF3D7_0519800 conserved protein, unknown function 3 9.1 6 PF3D7_0510200 peptidyl-prolyl cis-trans isomerase (CYP87) 4 9 4 PF3D7_1449500 transcription factor with AP2 domain(s), 4 9 3 154

putative (ApiAP2) PF3D7_1105600 translocon component PTEX88 (PTEX88 4 8.9 3 pre-mRNA-processing ATP-dependent RNA PF3D7_0508700 helicase PRP5, putative (PRP5) 16 8.7 19 conserved Plasmodium protein, unknown PF3D7_1419000 function 8 8.7 6 structural maintenance of chromosome PF3D7_1130700 protein, putative 9 8.6 11 PF3D7_1216900 DNA-binding chaperone, putative 6 8.6 10 ATP-dependent RNA helicase DDX23, PF3D7_0518500 putative (DDX23) 6 8.5 12 PF3D7_0515000 RNA-binding protein, putative 4 8.5 5 PF3D7_0623100 coronin binding protein, putative 4 8.5 4 large subunit rRNA processing protein, PF3D7_1405800 putative 5 8.4 14 protein arginine N-methyltransferase 5, PF3D7_1361000 putative (PRMT5) 3 8.4 5 Plasmodium exported protein, unknown PF3D7_0113200 function 3 8.3 3 PF3D7_1324200 micro-fibrillar-associated protein, putative 3 8.3 0 PF3D7_0106300 calcium-transporting ATPase (ATP6) 6 8.1 9 PF3D7_1432500 RNA methyltransferase, putative 3 7.9 2 conserved Plasmodium protein, unknown PF3D7_0218200 function 4 7.7 3 conserved Plasmodium protein, unknown PF3D7_0105800 function 4 7.6 2 1 origin recognition complex subunit 1 PF3D7_1203000 (ORC1) 6 7.5 3 PF3D7_1118200 heat shock protein 90, putative 4 7.5 15 PF3D7_1149200 ring-infected erythrocyte surface antigen 10 7.4 7 PF3D7_0707400 AAA family ATPase, putative 4 7.4 10 PF3D7_0624600 SNF2 helicase, putative (ISWI) 13 7.2 31 PF3D7_1025000 formin 2, putative 6 7.2 17 PF3D7_0731600;PF3D7 _0215300 acyl-CoA synthetase (ACS5) 3 7.2 5 conserved Plasmodium protein, unknown PF3D7_0927600 function 3 7.2 0 conserved Plasmodium protein, unknown PF3D7_0827300 function 3 7.1 0 conserved Plasmodium protein, unknown PF3D7_0111800 function 3 6.8 6 mature parasite-infected erythrocyte surface antigen,erythrocyte membrane PF3D7_0500800 protein 2 (MESA) 6 6.7 27 PF3D7_0630900 ATP-dependent RNA helicase HAS1 (HAS1) 3 6.7 7 conserved Plasmodium protein, unknown PF3D7_1126500 function 5 6.6 6 conserved Plasmodium protein, unknown PF3D7_1140800 function 4 6.6 0 155

conserved Plasmodium protein, unknown PF3D7_1124300 function 3 6.5 3 transcription factor with AP2 domain(s) PF3D7_1007700 (ApiAP2) 9 6.3 8 PF3D7_0529400.1;PF3D conserved Plasmodium protein, unknown 7_0529400.2 function 7 6.3 1 CCR4-NOT transcription complex subunit 4, PF3D7_1235300 putative (NOT4) 6 6.2 10 PF3D7_0903400 DEAD/DEAH box helicase, putative 9 6.1 13 U3 snoRNA-associated small subunit rRNA PF3D7_1333600 processing associated protein, putative 4 6.1 5 PF3D7_0801800 mannose-6-phosphate isomerase, putative 4 6 12 splicing factor 3B subunit 3, putative PF3D7_1234800 (SF3B3) 4 5.9 5 eukaryotic translation initation factor 4 PF3D7_1312900 gamma, putative (EIF4G) 4 5.6 5 conserved Plasmodium protein, unknown PF3D7_1127800 function 4 5.3 16 PF3D7_0622800 leucine--tRNA ligase, putative 4 5.3 8 eukaryotic translation initiation factor 5, PF3D7_1206700 putative 3 5.3 4 PF3D7_0606100 RNA-binding protein, putative 4 5.2 6 PF3D7_0804000 cactin homolog, putative 3 5.2 2

156

Appendix III: MyoB-BirA* and MTIPB-BirA* mass spectrometry data

MyoB-BirA* Sample

MS/ Sequence Protein IDs Fasta headers Peptides MS coverage Count

GTP-binding nuclear protein RAN/TC4 PF3D7_1117700 (RAN) 18 86.4 37 PF3D7_0306800 T-complex protein beta subunit, putative 39 85 58 glyceraldehyde-3-phosphate PF3D7_1462800 dehydrogenase (GAPDH) 21 80.4 47 PF3D7_1357100;PF3D 7_1357000 elongation factor 1-alpha 37 80.1 147 PF3D7_0922500 phosphoglycerate kinase (PGK) 35 79.3 59 conserved Plasmodium protein, unknown PF3D7_1213200 function 26 77.7 56 PF3D7_0818200 14-3-3 protein (14-3-3I) 19 77.5 29 PF3D7_0626800 pyruvate kinase (PyrK) 32 77.3 73 PF3D7_1246200 actin I (ACT1) 28 76.6 43 PF3D7_1016300;PF3D 7_0937000 glycophorin binding protein (GBP) 26 75.4 67 PF3D7_0708400 heat shock protein 90 (HSP90) 84 75 586 PF3D7_1008700 tubulin beta chain 23 73.9 32 PF3D7_0503600 myosin B (MyoB) 65 73.8 126 PF3D7_1446600 centrin-2 (CEN2) 8 73.2 11 PF3D7_0722400 GTP-binding protein, putative 20 72.8 29 PF3D7_0922200 S-adenosylmethionine synthetase (SAMS) 25 71.1 41 PF3D7_1444800 fructose-bisphosphate aldolase (FBPA) 24 70.7 35 phosphoethanolamine N- PF3D7_1343000 methyltransferase (PMT) 16 70.7 41 PF3D7_0516200 40S ribosomal protein S11 8 70.2 9 PF3D7_1451100 elongation factor 2 53 70 85 PF3D7_0624000 hexokinase (HK) 33 68.6 47 PF3D7_0322900 40S ribosomal protein S3A, putative 23 68.3 33 PF3D7_1105000 histone H4 (H4) 14 68 31 high molecular weight rhoptry protein 2 PF3D7_0929400 (RhopH2) 97 67.8 146 phosphoglycerate mutase, putative PF3D7_1120100 (PGM1) 14 67.6 18 PF3D7_0416800 small GTP-binding protein sar1 (SAR1) 11 67.2 17 157

conserved Plasmodium protein, unknown PF3D7_0520200 function 44 67 75 PF3D7_0608800 ornithine aminotransferase (OAT) 25 66.7 39 PF3D7_1426000 60S ribosomal protein L21 (RPL21) 13 66.5 20 PF3D7_1124600 ethanolamine kinase (EK) 28 66.2 38 PF3D7_1468700 eukaryotic initiation factor 4A (eIF4A) 27 65.8 32 PF3D7_0316800 40S ribosomal protein S15A, putative 10 65.4 13 PF3D7_0818900;PF3D 7_0831700 heat shock protein 70 (HSP70) 56 65.3 179 ATP-dependent RNA helicase UAP56 PF3D7_0209800 (UAP56) 25 65 40 PF3D7_1026800 40S ribosomal protein S2 (RPS2) 12 65 15 PF3D7_1108400 casein kinase 2, alpha subunit (CK2alpha) 18 64.8 26 PF3D7_0422400 40S ribosomal protein S19 (RPS19) 11 64.7 15 PF3D7_1020900 ADP-ribosylation factor (ARF1) 8 64.1 15 PF3D7_1105400 40S ribosomal protein S4, putative 18 63.6 34 PF3D7_0524000 karyopherin beta (KASbeta) 57 63.4 91 PF3D7_1224000 GTP cyclohydrolase I (GCH1) 28 62.7 45 PF3D7_1357800 TCP-1/cpn60 chaperonin family, putative 23 62.6 27 PF3D7_1015900 enolase (ENO) 20 62.6 28 T-complex protein 1, gamma subunit, PF3D7_1229500 putative 29 62 35 conserved Plasmodium protein, unknown PF3D7_1205600 function 26 61.9 39 hypoxanthine-guanine PF3D7_1012400 phosphoribosyltransferase (HGPRT) 12 61.9 18 PF3D7_1338200 60S ribosomal protein L6-2, putative 13 61.5 19 PF3D7_0308200 TCP-1/cpn60 chaperonin family, putative 20 60.5 30 haloacid dehalogenase-like hydrolase PF3D7_1033400 (HAD1) 10 60.4 13 PF3D7_1460700 60S ribosomal protein L27 (RPL27) 11 60.3 14 40S ribosomal protein S11, putative PF3D7_0317600 (RPS11) 13 60.2 18 PF3D7_0826700 receptor for activated c kinase (RACK) 14 60.1 18 PF3D7_1465900 40S ribosomal protein S3 17 59.7 28 PF3D7_0610400 histone H3 (H3) 8 59.6 16 PF3D7_1011800 PRE-binding protein (PREBP) 77 59.4 127 PF3D7_0406100 vacuolar ATP synthase subunit b 19 59.3 29 PF3D7_1019400 60S ribosomal protein L30e, putative 6 59.3 7 PF3D7_0507100 60S ribosomal protein L4 (RPL4) 29 59.1 63 PF3D7_0618300 60S ribosomal protein L27a, putative 11 58.8 14 PF3D7_1347500 DNA/RNA-binding protein Alba 4 (ALBA4) 21 58.6 23 PF3D7_1008800 nucleolar protein 5, putative (NOP5) 21 58.6 23 PF3D7_1311900 vacuolar ATP synthase subunit a (vapA) 25 58.3 24 PF3D7_0721600 40S ribosomal protein S5, putative 12 57.9 16 PF3D7_0617800 histone H2A (H2A) 5 57.6 16 158

conserved Plasmodium protein, unknown PF3D7_1118700 function 38 57.5 51 PF3D7_1414300 60S ribosomal protein L10, putative 16 57.5 36 PF3D7_1323100 60S ribosomal protein L6, putative 9 57.4 11 PF3D7_1136500.1;PF3 D7_1136500.2 casein kinase 1 (CK1) 17 57.3 22 PF3D7_0110400 DNA-directed RNA polymerase 2, putative 16 57 28 PF3D7_1331800 60S ribosomal protein L23, putative 8 56.8 14 PF3D7_1447000 40S ribosomal protein S5 13 56.6 23 PF3D7_1238100 calcyclin binding protein, putative 11 56.6 11 glutathione peroxidase-like thioredoxin PF3D7_1212000 peroxidase (TPx(Gl)) 11 56.6 11 multiprotein bridging factor type 1, PF3D7_1128200 putative (MBF1) 8 56.6 8 conserved Plasmodium protein, unknown PF3D7_1363600 function 10 56.4 15 PF3D7_1362200 RuvB-like helicase 3 (RUVB3) 19 56.3 21 PF3D7_1027800 60S ribosomal protein L3 (RPL3) 26 56.2 48 PF3D7_0510500 topoisomerase I (TopoI) 47 56.1 81 cytoadherence linked asexual protein 3.1 PF3D7_0302500 (CLAG3.1) 73 56 105 PF3D7_1130200 60S ribosomal protein P0 (PfP0) 12 56 15 PF3D7_1202900 high mobility group protein B1 (HMGB1) 5 55.7 7 PF3D7_1341200 60S ribosomal protein L18, putative 15 55.4 20 PF3D7_1302800 40S ribosomal protein S7, putative 12 55.2 22 PF3D7_1424100 60S ribosomal protein L5, putative 17 55.1 28 26S protease regulatory subunit 7, PF3D7_1311500 putative (RPT1) 16 55 23 T-complex protein 1 epsilon subunit, PF3D7_0320300 putative 26 54.8 28 PF3D7_0807300 ras-related protein Rab-18 (RAB18) 8 54.7 10 PF3D7_0717700 serine--tRNA ligase, putative 20 54.4 20 PF3D7_0615400 ribonuclease, putative 155 54.3 224 PF3D7_1324900 L-lactate dehydrogenase (LDH) 11 54.1 14 PF3D7_0503400 actin-depolymerizing factor 1 (ADF1) 5 54.1 9 PF3D7_1438900 thioredoxin peroxidase 1 (Trx-Px1) 7 53.8 10 PF3D7_0915400 6-phosphofructokinase (PFK9) 58 53.5 75 conserved Plasmodium protein, unknown PF3D7_1105800 function 11 53.4 11 PF3D7_0813900 40S ribosomal protein S16, putative 10 52.8 14 conserved Plasmodium protein, unknown PF3D7_1457300 function 23 52.7 28 PF3D7_1126200 40S ribosomal protein S18, putative 7 52.6 6 PF3D7_0903200 ras-related protein RAB7 (RAB7) 7 52.4 9 PF3D7_1408600 40S ribosomal protein S8e, putative 11 52.3 19 eukaryotic translation initiation factor 2 PF3D7_1010600 beta subunit, putative 11 52.3 12 159

high molecular weight rhoptry protein 3 PF3D7_0905400 (RhopH3) 35 52.2 57 PF3D7_0303200 HAD superfamily protein, putative 65 51.9 107 inner membrane complex sub- PF3D7_1460600 compartment protein 3, putative (ISP3) 5 51.4 5 endoplasmic reticulum-resident calcium PF3D7_1108600 binding protein (ERC) 18 51.3 29 nuclear movement protein, putative PF3D7_1336800 (NUDC) 17 51.3 20 box C/D snoRNP rRNA 2-O-methylation PF3D7_1118500 factor, putative 30 51 46 PF3D7_1317800 40S ribosomal protein S19 (RPS19) 7 51 17 PF3D7_1437900 HSP40, subfamily A, putative 15 50.9 21 PF3D7_1136300 tudor staphylococcal nuclease (TSN) 56 50.8 64 PF3D7_0823800 DnaJ protein, putative 27 50.8 34 PF3D7_0917900 heat shock protein 70 (HSP70-2) 31 50.5 42 PF3D7_0903700 alpha tubulin 1 17 50.3 22 PF3D7_0501600 rhoptry-associated protein 2 (RAP2) 16 50.3 16 PF3D7_0706400 60S ribosomal protein L37 (RPL37) 6 50 6 conserved Plasmodium protein, unknown PF3D7_0628600 function 19 49.9 24 PF3D7_0608700 chaperone, putative 22 49.7 25 PF3D7_1342600 myosin A (MyoA) 26 49.4 28 PF3D7_0617900 histone H3 variant, putative (H3.3) 3 49.3 7 PF3D7_0709700;PF3D 7_0102400 lysophospholipase, putative 12 49.2 16 cAMP-dependent protein kinase catalytic PF3D7_0934800 subunit (PKAc) 17 49.1 22 PF3D7_0517000 60S ribosomal protein L12, putative 6 49.1 4 PF3D7_1225800 ubiquitin-activating enzyme E1 (UBA1) 35 49 43 26S protease regulatory subunit 10B, PF3D7_1306400 putative (RPT4) 14 48.9 15 PF3D7_1034900 methionine--tRNA ligase, putative 39 48.8 48 PF3D7_1360900 polyadenylate-binding protein, putative 11 48.8 11 PF3D7_1027700.2;PF3 D7_1027700.1 centrin-3 (CEN3) 6 48.6 7 PF3D7_0814000 60S ribosomal protein L13-2, putative 13 48.4 18 PF3D7_1006800 RNA-binding protein, putative 11 48.4 17 signal recognition particle subunit SRP54 PF3D7_1450100 (SRP54) 18 48.2 19 PF3D7_1242700 40S ribosomal protein S17, putative 7 48.2 9 PF3D7_1424400 60S ribosomal protein L7-3, putative 20 48.1 28 6-phosphogluconate dehydrogenase, PF3D7_1454700 decarboxylating, putative 16 47.9 16 PF3D7_1421200 40S ribosomal protein S25 (RPS25) 6 47.6 6 PF3D7_1308300 40S ribosomal protein S27 (RPS27) 4 47.6 5 ADP/ATP transporter on adenylate PF3D7_1037300 translocase (ADT) 14 47.5 16 160

PF3D7_0307200 60S ribosomal protein L7, putative 13 46.7 23 conserved Plasmodium protein, unknown PF3D7_1002900 function 4 46.5 5 rRNA-processing protein EBP2, putative PF3D7_1028300 (EBP2) 14 46.3 17 PF3D7_1472200 histone deacetylase, putative (HDA1) 110 46.2 139 PF3D7_1138500 protein phosphatase 2C (PP2C) 32 46.2 44 conserved Plasmodium protein, unknown PF3D7_0411100 function 5 45.9 6 PF3D7_0321600 ATP-dependent RNA helicase, putative 22 45.4 29 PF3D7_1358700 HVA22-like protein, putative 5 45.1 4 conserved Plasmodium protein, unknown PF3D7_0522000 function 3 44.9 3 PF3D7_0708800 heat shock protein 110 (HSP110c) 27 44.8 36 conserved Plasmodium protein, unknown PF3D7_0513200 function 111 44.7 138 H/ACA ribonucleoprotein complex subunit PF3D7_1417500 4, putative (CBF5) 13 44.6 14 PF3D7_0516900 60S ribosomal protein L2 (RPL2) 11 44.6 18 conserved Plasmodium protein, unknown PF3D7_0306100 function 18 44.4 22 eukaryotic translation initiation factor 3 PF3D7_0612100 subunit L, putative 20 44.2 26 PF3D7_1302100 gamete antigen 27/25 (Pfg27) 6 44.2 13 conserved Plasmodium protein, unknown PF3D7_1466800 function 34 44.1 46 S-adenosyl-L-homocysteine hydrolase PF3D7_0520900 (SAHH) 24 44.1 39 PF3D7_0802200 1-cys peroxiredoxin (1-CysPxn) 7 44.1 9 26S proteasome AAA-ATPase subunit PF3D7_0413600 RPT3, putative 12 43.9 11 PF3D7_1342000 40S ribosomal protein S6 24 43.8 76 cytoadherence linked asexual protein 9 PF3D7_0935800 (CLAG9) 53 43.6 66 PF3D7_1025000 formin 2, putative 44 43.6 70 PF3D7_1349200 glutamate--tRNA ligase, putative 30 43.5 34 ATP-dependent RNA helicase DDX5, PF3D7_1445900 putative (DDX5) 17 43.5 19 PF3D7_0814200 DNA/RNA-binding protein Alba 1 (ALBA1) 12 43.5 17 PF3D7_0512600 ras-related protein Rab-1B (RAB1b) 7 43.5 7 PF3D7_0903900 60S ribosomal protein L32 (RPL32) 5 43.5 7 PF3D7_0214000 T-complex protein 1, putative 21 43.4 22 PF3D7_1420600 pantothenate kinase, putative (PANK) 17 43.2 19 eukaryotic translation initiation factor 2 PF3D7_1410600 gamma subunit, putative 14 42.8 18 cAMP-dependent protein kinase PF3D7_1223100 regulatory subunit (PKAr) 20 42.6 26 PF3D7_1308200 carbamoyl phosphate synthetase (cpsSII) 66 42.5 76 161

PF3D7_1222300 endoplasmin, putative (GRP94) 25 42.4 30 PF3D7_0612900 nucleolar GTP-binding protein 1, putative 20 42.3 21 conserved Plasmodium protein, unknown PF3D7_0828300 function 24 42.2 41 heat shock protein DNAJ homologue Pfj4 PF3D7_1211400 (PfJ4) 9 42.2 10 PF3D7_1407100 fibrillarin, putative (NOP1) 13 42.1 16 conserved Plasmodium protein, unknown PF3D7_0911200 function 7 42.1 6 PF3D7_0312800 60S ribosomal protein L26, putative 5 42.1 6 PF3D7_0821700 60S ribosomal protein L22, putative 7 41.7 11 PF3D7_1332900 isoleucine--tRNA ligase, putative 39 41.5 46 PF3D7_0933200 calcyclin binding protein, putative 7 41.5 7 PF3D7_1410400 rhoptry-associated protein 1 (RAP1) 24 41.4 26 conserved Plasmodium membrane PF3D7_1237700 protein, unknown function 7 41.4 8 PF3D7_1130100 60S ribosomal protein L38 (RPL38) 5 41.4 6 lysine-rich membrane-associated PHISTb PF3D7_0532400 protein (LyMP) 16 41.3 20 PF3D7_1338300 elongation factor 1-gamma, putative 20 41.1 25 PF3D7_1105100 histone H2B (H2B) 8 41 34 ribonucleotide reductase small subunit, PF3D7_1015800 putative 14 40.9 20 conserved Plasmodium protein, unknown PF3D7_0710200 function 105 40.8 117 PF3D7_0827900 protein disulfide isomerase (PDI8) 11 40.8 12 PF3D7_1336400 RanBPM and CLTH-like protein, putative 7 40.8 8 PF3D7_0817700 rhoptry neck protein 5 (RON5) 39 40.7 46 PF3D7_1003500 40S ribosomal protein S20e, putative 7 40.7 10 PF3D7_0606900 glutaredoxin-like protein (GLP2) 5 40.6 6 PF3D7_1358800 40S ribosomal protein S15 (RPS15) 8 40.4 9 conserved Plasmodium protein, unknown PF3D7_1361800 function 71 40.2 84 conserved Plasmodium protein, unknown PF3D7_1359600 function 54 40.1 66 conserved Plasmodium protein, unknown PF3D7_1004500 function 7 39.6 10 PF3D7_1252100 rhoptry neck protein 3 (RON3) 69 39.5 89 PF3D7_1434300 Hsp70/Hsp90 organizing protein (HOP) 14 39.4 14 PF3D7_0415900 60S ribosomal protein L15, putative 10 39 14 PF3D7_1139900 conserved protein, unknown function 6 38.9 8 proliferation-associated protein 2g4, PF3D7_1428300 putative 17 38.7 18 PF3D7_1142500 60S ribosomal protein L28 (RPL28) 5 38.6 6 PF3D7_1340300 nucleolar complex protein 2, putative 38 38.4 67 eukaryotic translation initiation factor 3 PF3D7_0918300 subunit 5, putative 7 38.4 8 PF3D7_1459400 conserved Plasmodium protein, unknown 12 38.2 15 162

function PF3D7_0511800 inositol-3-phosphate synthase (INO1) 18 38.1 16 PF3D7_0706000 importin-7, putative 38 37.8 43 replication protein A1, small fragment PF3D7_0904800 (RPA1) 16 37.8 20 PF3D7_0812400 karyopherin alpha (KARalpha) 15 37.8 22 PF3D7_1213800 proline--tRNA ligase (PRS) 22 37.7 26 PF3D7_0525100 acyl-CoA synthetase (ACS10) 17 37.6 18 26S protease regulatory subunit 6a, PF3D7_1130400 putative (RPT5) 12 37.6 15 ubiquitin domain-containing protein PF3D7_1113400 DSK2, putative (DSK2) 9 37.6 10 PF3D7_0719600 60S ribosomal protein L11a, putative 6 37.6 8 PF3D7_0716600 cysteine desulfurase (SufS) 16 37.5 23 conserved Plasmodium protein, unknown PF3D7_0813300 function 12 37.5 18 PF3D7_0111500 UMP-CMP kinase, putative 11 37.5 10 PF3D7_1323200 vacuolar ATP synthase subunit g, putative 6 37.4 5 eukaryotic translation initiation factor 3, PF3D7_0528200 subunit 6, putative 14 37.3 13 PF3D7_0320900 histone H2A variant, putative (H2A.Z) 4 37.3 6 PF3D7_1027300 peroxiredoxin (nPrx) 26 37.2 58 PF3D7_0919000 nucleosome assembly protein (NAPS) 7 37.2 8 26S protease regulatory subunit 4, PF3D7_1008400 putative (RPT2) 10 37.1 10 signal recognition particle subunit SRP72, PF3D7_1136400 putative (SRP72) 28 37 35 PF3D7_0929200 RNA-binding protein, putative 7 37 9 PF3D7_1018500 PHF5-like protein, putative 3 36.9 3 PF3D7_0627700 transportin 36 36.8 40 PF3D7_1365900;PF3D 7_1211800 ubiquitin-60S ribosomal protein L40 4 36.7 9 PF3D7_1352500 thioredoxin-related protein, putative 7 36.5 9 PF3D7_0210100.1 60S ribosomal protein L37ae, putative 4 36.5 4 PF3D7_1309100 60S ribosomal protein L24, putative 8 36.4 8 conserved Plasmodium protein, unknown PF3D7_1460500 function 42 36.3 47 DNA replication licensing factor MCM3, PF3D7_0527000 putative (MCM3) 25 36.1 26 PF3D7_1116200.1;PF3 pyridoxine biosynthesis protein PDX2 D7_1116200.2 (PDX2) 5 36.1 5 PF3D7_1020700 histone acetyltransferase, putative 35 36 36 PF3D7_1108700 heat shock protein DnaJ homologue Pfj2 17 35.9 22 PF3D7_1145400 dynamin-like protein (DYN1) 19 35.8 22 PF3D7_0413700 lysine decarboxylase-like protein, putative 8 35.8 7 splicing factor 3A subunit 1, putative PF3D7_1474500 (SF3A1) 17 35.4 15 PF3D7_1444300 1-acyl-sn-glycerol-3-phosphate 6 35.3 8 163

acyltransferase, putative (LPAAT) mannose-6-phosphate isomerase, PF3D7_0801800 putative 27 35.2 32 PF3D7_1129400 RNA methyltransferase, putative 24 35.2 39 PF3D7_0526200.2;PF3 ADP-ribosylation factor GTPase-activating D7_0526200.1 protein, putative (ARF-GAP) 12 35.2 11 conserved Plasmodium protein (10b PF3D7_1021900 antigen), unknown function 75 35.1 100 PF3D7_1426100 basic transcription factor 3b, putative 6 35.1 8 PF3D7_0714000 histone H2B variant (H2B.Z) 6 35 9 PF3D7_0627500 protein DJ-1 (DJ1) 4 34.9 4 PF3D7_0930300;PF3D 7_1312000 merozoite surface protein 1 (MSP1) 50 34.8 54 PF3D7_0419600 ran binding protein 1, putative 13 34.6 16 PF3D7_0304400 60S ribosomal protein L44 (RPL44) 10 34.6 13 rab specific GDP dissociation inhibitor PF3D7_1242800 (rabGDI) 10 34.6 12 polyadenylate-binding protein, putative PF3D7_1224300 (PABP) 24 34.5 30 PF3D7_1217900 conserved protein, unknown function 10 34.2 14 PF3D7_1239200;REV_ transcription factor with AP2 domain(s) _PF3D7_1348400 (ApiAP2) 55 34.1 65 eukaryotic translation initiation factor, PF3D7_0815600 putative 5 34 6 PF3D7_0315100 translation initiation factor 4E (eIF4E) 5 33.9 6 PF3D7_1368200 RNAse L inhibitor protein, putative 16 33.4 18 PF3D7_1104400 conserved protein, unknown function 14 33.3 17 conserved Plasmodium protein, unknown PF3D7_0710700 function 11 33.3 18 PF3D7_1231100 ras-related protein Rab-2 (RAB2) 5 33.3 6 PF3D7_1203700 nucleosome assembly protein (NAPL) 12 33.1 12 signal recognition particle receptor, beta PF3D7_1246800 subunit (SRPR-beta) 9 33 8 conserved Plasmodium protein, unknown PF3D7_1422400 function 46 32.9 59 eukaryotic translation initiation factor 3 PF3D7_1206200 subunit 8, putative 31 32.9 40 mediator of RNA polymerase II PF3D7_0822100 transcription subunit 7, putative (MED7) 6 32.9 6 M1-family alanyl aminopeptidase PF3D7_1311800 (M1AAP) 22 32.8 21 conserved Plasmodium protein, unknown PF3D7_1419700 function 12 32.8 22 ribonucleotide reductase small subunit PF3D7_1405600 (RNR) 10 32.7 14 Leu/Phe-tRNA protein transferase, PF3D7_0212900 putative 8 32.6 7 conserved Plasmodium protein, unknown PF3D7_0510100 function 54 32.4 62 164

PF3D7_0306900 40S ribosomal protein S23, putative 3 32.4 5 PF3D7_0211800 asparagine--tRNA ligase (AsnRS) 12 32.3 17 PF3D7_0520000 40S ribosomal protein S9, putative 6 32.3 8 PF3D7_0810800 dihydropteroate synthetase (DHPS) 16 32.2 18 PF3D7_1218600 arginine--tRNA ligase, putative 14 32.2 13 PF3D7_0607000 translation initiation factor IF-2, putative 23 32.1 24 PF3D7_1341300 60S ribosomal protein L18-2, putative 5 32.1 8 conserved Plasmodium protein, unknown PF3D7_1210300 function 4 32.1 6 PF3D7_1351400 60S ribosomal protein L17, putative 7 32 15 PF3D7_1412500 actin II (ACT2) 7 31.9 7 PF3D7_1464700 ATP synthase (C/AC39) subunit, putative 7 31.9 7 PF3D7_0934500 vacuolar ATP synthase subunit e, putative 4 31.9 5 PF3D7_0106800 ras-related protein Rab-5C (RAB5c) 5 31.8 5 PF3D7_0217800 40S ribosomal protein S26 (RPS26) 4 31.8 4 golgi organization and biogenesis factor, PF3D7_0418200 putative 11 31.7 12 PF3D7_0705700 40S ribosomal protein S29, putative 3 31.5 3 lysine-specific histone demethylase 1, PF3D7_1211600 putative (LSD1) 73 31.3 79 signal recognition particle subunit SRP68, PF3D7_0621900 putative (SRP68) 17 31.3 21 PF3D7_0710600 60S ribosomal protein L34 (RPL34) 7 31.3 30 PF3D7_1029600 adenosine deaminase (ADA) 6 31.3 6 PF3D7_1460300 60S ribosomal protein L29, putative 3 31.3 11 PF3D7_1323400 60S ribosomal protein L23 (RPL23) 11 31.1 22 autophagy-related protein 18, putative PF3D7_1012900 (ATG18) 10 31.1 10 PF3D7_1471100 exported protein 2 (EXP2) 6 31 6 PF3D7_0107000 centrin-1 (CEN1) 5 31 7 serine/threonine protein phosphatase PF3D7_1414400 PP1 (PP1) 6 30.9 7 PF3D7_0527500 Hsc70-interacting protein (HIP) 13 30.8 16 PF3D7_0719700 40S ribosomal protein S10, putative 4 30.7 6 conserved Plasmodium protein, unknown PF3D7_1226400 function 19 30.6 24 PF3D7_1134000 heat shock protein 70 (HSP70-3) 17 30.6 19 PF3D7_0102900 aspartate--tRNA ligase 15 30.4 17 PF3D7_1410200 cytidine triphosphate synthetase 19 30.2 18 FACT complex subunit SSRP1, putative PF3D7_1441400 (FACT-S) 13 30.2 15 conserved Plasmodium protein, unknown PF3D7_0821000 function 10 30.2 13

choline/ethanolaminephosphotransferase, PF3D7_0628300 putative (CEPT) 8 30.2 9 PF3D7_1469800 conserved Plasmodium protein, unknown 7 30.2 8 165

function conserved Plasmodium protein, unknown PF3D7_0108300 function 51 29.9 59 deoxyribodipyrimidine photo-lyase, PF3D7_0513600 putative 26 29.6 29 ribonucleoside-diphosphate reductase, PF3D7_1437200 large subunit, putative 18 29.6 20 conserved Plasmodium protein, unknown PF3D7_0308100 function 38 29.5 43 PF3D7_0807900 tyrosine--tRNA ligase (TyrRS) 8 29.5 7 eukaryotic translation initiation factor 3 PF3D7_0716800 37.28 kDa subunit, putative 7 29.4 9 eukaryotic translation initiation factor 3 PF3D7_1212700 subunit 10, putative 37 29.3 40 PF3D7_0519400 40S ribosomal protein S24 (RPS24) 4 29.3 4 PF3D7_1420400 glycine--tRNA ligase (GlyRS) 18 29.2 22 PF3D7_0518600 WD-repeat protein, putative 31 29 37 PF3D7_0505800 small ubiquitin-related modifier (SUMO) 3 29 4 conserved Plasmodium protein, unknown PF3D7_1412100 function 18 28.9 18 ubiquitin-40S ribosomal protein S27a, PF3D7_1402500 putative 8 28.9 14 PF3D7_0918000 secreted acid phosphatase (GAP50) 9 28.8 10 PF3D7_1438700 DNA primase small subunit 9 28.8 9 succinate dehydrogenase subunit 4, PF3D7_1010300 putative (SDH4) 4 28.8 4 PF3D7_1021500 RNA helicase, putative 16 28.7 16 PF3D7_0414200.1;PF3 D7_0414200.2 calmodulin-like protein 4 28.7 4 conserved Plasmodium protein, unknown PF3D7_1446700 function 5 28.3 5 PF3D7_0815200 importin beta, putative 20 28.2 22 PF3D7_1401800 choline kinase (CK) 10 28.2 13 PF3D7_1004000 60S ribosomal protein L13, putative 8 28.2 10 26S proteasome regulatory subunit PF3D7_0312300 RPN12, putative (RPN12) 4 28 4 conserved Plasmodium protein, unknown PF3D7_1323900 function 8 27.9 13 PF3D7_0218000 replication factor C subunit 2, putative 6 27.9 6 PF3D7_0321500 peptidase, putative 25 27.8 32 PF3D7_1336900 tryptophan--tRNA ligase (WRS) 11 27.8 12 PF3D7_0512700 orotate phosphoribosyltransferase (OPRT) 6 27.8 5 eukaryotic translation initiation factor 2 PF3D7_0728000 alpha subunit, putative 7 27.7 6 conserved Plasmodium protein, unknown PF3D7_1006700 function 6 27.7 7 PF3D7_1109900 60S ribosomal protein L36 (RPL36) 4 27.7 5 conserved Plasmodium protein, unknown PF3D7_1014900 function 58 27.6 70 166

PF3D7_1452000 rhoptry neck protein 2 (RON2) 49 27.6 60 conserved Plasmodium protein, unknown PF3D7_1230800 function 12 27.6 12 PF3D7_1441200 60S ribosomal protein L1, putative 5 27.6 6 PF3D7_1417800;PF3D DNA replication licensing factor MCM2 7_0525600 (MCM2) 20 27.4 22 PF3D7_1201500 XPA binding protein 1, putative 8 27.4 9 PF3D7_0106300 calcium-transporting ATPase (ATP6) 22 27.3 29 eukaryotic translation initiation factor 5A PF3D7_1204300 (EIF5A) 3 27.3 5 conserved Plasmodium protein, unknown PF3D7_0932800 function 26 27.2 29 eukaryotic translation initiation factor, PF3D7_0517700 putative 12 27.2 11 PF3D7_0422700 eukaryotic initiation factor, putative 7 27.2 7 NLI interacting factor-like phosphatase, PF3D7_1012700 putative (NIF4) 27 27.1 29 PF3D7_1132200 TCP-1/cpn60 chaperonin family, putative 10 27 10 PF3D7_0302900 exportin-1, putative 27 26.9 29 protein geranylgeranyltransferase type II, PF3D7_1242600 alpha subunit, putative 10 26.9 11 phosphoenolpyruvate carboxykinase PF3D7_1342800 (PEPCK) 10 26.9 9 PF3D7_0624600 SNF2 helicase, putative (ISWI) 58 26.8 67 glideosome associated protein with PF3D7_1406800 multiple membrane spans 3 (GAPM3) 5 26.8 6 conserved Plasmodium protein, unknown PF3D7_1468100 function 46 26.7 52 conserved Plasmodium protein, unknown PF3D7_1026000 function 8 26.7 10 PF3D7_1463200 replication factor C3, putative (RFC3) 6 26.7 5 PF3D7_1219100 clathrin heavy chain, putative 37 26.6 43 PF3D7_1134100;PF3D 7_0710000 protein disulfide isomerase (PDI-11) 9 26.5 11 26S proteasome regulatory subunit RPN3, PF3D7_1338100 putative (RPN3) 11 26.4 8 haloacid dehalogenase-like hydrolase, PF3D7_1226300 putative (HAD2) 5 26.4 6 U1 small nuclear ribonucleoprotein A, PF3D7_1306900 putative 8 26.3 7 PF3D7_1350100 lysine--tRNA ligase (KRS1) 11 26.2 13 PF3D7_1422800 actin-related protein, putative (ARP4a) 10 26.2 13 conserved Plasmodium protein, unknown PF3D7_0813500 function 10 26.2 9 subunit of proteaseome activator PF3D7_0907700 complex, putative 5 26.2 5 trafficking protein particle complex PF3D7_0310700 subunit 4, putative 3 26.2 3 PF3D7_0312400 glycogen synthase kinase 3 (GSK3) 8 26.1 9 167

U3/U14 snoRNA-associated small subunit PF3D7_1109400 rRNA processing protein, putative 8 26.1 7 PF3D7_0206700 adenylosuccinate lyase (ASL) 8 26.1 7 conserved Plasmodium protein, unknown PF3D7_1241100 function 7 26.1 7 vesicle-associated membrane protein, PF3D7_1439800 putative 4 26.1 7 nascent polypeptide associated complex PF3D7_0621800 alpha chain, putative 4 26.1 4 PF3D7_1405900 RNA-binding protein, putative 26 25.9 28 conserved Plasmodium protein, unknown PF3D7_1356100 function 18 25.9 18 conserved Plasmodium protein, unknown PF3D7_1411500 function 19 25.8 24 vacuolar protein sorting-associated PF3D7_1406700 protein 29, putative (VPS29) 3 25.8 3 large subunit rRNA methyltransferase, PF3D7_1354300 putative 24 25.7 27 PF3D7_1318800 translocation protein SEC63 (SEC63) 14 25.7 12 conserved Plasmodium protein, unknown PF3D7_1210600 function 24 25.6 25 PF3D7_1118200 heat shock protein 90, putative 16 25.6 13 regulator of chromosome condensation, PF3D7_0711500 putative 9 25.6 11 conserved Plasmodium membrane PF3D7_1409400 protein, unknown function 8 25.6 11 PF3D7_1458400 aminodeoxychorismate lyase (ADCL) 7 25.6 8 conserved Plasmodium protein, unknown PF3D7_1238700 function 9 25.5 10 choline-phosphate cytidylyltransferase PF3D7_1316600 (CCT) 12 25.4 12 PF3D7_0614500 60S ribosomal protein L19 (RPL19) 6 25.3 9 eukaryotic translation initiation factor PF3D7_1019000 subunit eIF2A, putative 49 25.2 60 PF3D7_0810600 RNA helicase, putative 17 25.2 17 PF3D7_0813200 CS domain protein, putative 5 25.2 6 26S proteasome regulatory subunit p55, PF3D7_1017900 putative (RPN5) 8 25.1 8 conserved Plasmodium protein, unknown PF3D7_0629600 function 6 25.1 5 PF3D7_1107400 DNA repair protein RAD51 (RAD51) 5 24.6 5 conserved Plasmodium protein, unknown PF3D7_1138700 function 39 24.5 41 conserved Plasmodium protein, unknown PF3D7_0217900 function 9 24.5 11 PF3D7_0106100 vacuolar ATP synthase subunit c, putative 8 24.5 7 PF3D7_1453700 co-chaperone p23 (P23) 6 24.4 11 PF3D7_0310600.2;PF3 SAC3/GNAP family-related protein, D7_0310600.1 putative 3 24.4 3 PF3D7_0316300.1;PF3 inorganic pyrophosphatase, putative 7 24.2 7 168

D7_0316300.2 PF3D7_0523000 multidrug resistance protein (MDR1) 25 24.1 25 PF3D7_0627800 acetyl-CoA synthetase, putative (ACS) 12 24.1 13 PF3D7_0213100 heat shock protein 40, putative 5 24.1 6 conserved Plasmodium protein, unknown PF3D7_0907300 function 5 24 6 PF3D7_1008900 adenylate kinase (AK1) 4 24 4 FACT complex subunit SPT16, putative PF3D7_0517400 (FACT-L) 21 23.9 19 DNA mismatch repair protein MSH2, PF3D7_1427500 putative (MSH2-1) 14 23.9 16 pyridoxine biosynthesis protein PDX1 PF3D7_0621200 (PDX1) 5 23.9 6 pre-mRNA-splicing factor SYF1, putative PF3D7_1235900 (XAB2) 16 23.7 16 conserved Plasmodium protein, unknown PF3D7_1456700 function 12 23.7 15 bifunctional dihydrofolate reductase- PF3D7_0417200 thymidylate synthase (DHFR-TS) 11 23.7 13 glycerol-3-phosphate dehydrogenase, PF3D7_1216200 putative 7 23.7 7 PF3D7_1431700 60S ribosomal protein L14, putative 4 23.6 5 PF3D7_0725000 exonuclease I, putative 19 23.3 23 PF3D7_0113000 glutamic acid-rich protein (GARP) 11 23.3 32 conserved Plasmodium protein, unknown PF3D7_0407700 function 26 23.1 23 PF3D7_1445200 RNA helicase, putative 14 23 18 cochaperone prefoldin complex subunit, PF3D7_1128100 putative 5 23 6 conserved Plasmodium protein, unknown PF3D7_0914400 function 3 22.9 3 ATP-dependent RNA helicase DBP10, PF3D7_0827000 putative (DBP10) 21 22.7 20 nicotinate phosphoribosyltransferase, PF3D7_0629100 putative (NAPRT) 11 22.7 10 cell differentiation protein, putative PF3D7_0507600 (CAF40) 9 22.7 11 26S proteasome regulatory subunit RPN6 PF3D7_1402300 (RPN6) 8 22.7 8 PF3D7_1241700 replication factor C subunit 4, putative 6 22.6 6 small subunit rRNA processing factor, PF3D7_1451900 putative 16 22.5 18 peptide chain release factor subunit 1, PF3D7_0212300 putative 7 22.5 8 conserved Plasmodium protein, unknown PF3D7_1447700 function 6 22.5 5 PF3D7_1145100 coatomer subunit gamma, putative 15 22.4 15 chromatin assembly factor 1 subunit, PF3D7_1329300 putative 10 22.3 12 PF3D7_1225500 small subunit rRNA processing factor, 7 22.3 8 169

putative PF3D7_1106000 RuvB-like helicase 2 (RUVB2) 7 22.3 7 PF3D7_1353400 Ran-binding protein, putative 19 22.2 23 cell division cycle protein 48 homologue, PF3D7_0619400 putative 13 22.2 15 PF3D7_0825500 protein KRI1, putative (KRI1) 10 22.2 11 conserved Plasmodium protein, unknown PF3D7_1306200 function 7 22.2 8 PF3D7_1205900 conserved protein, unknown function 16 22.1 21 DNA replication licensing factor MCM4 PF3D7_1317100 (MCM4) 14 22.1 16 PF3D7_0409800 zinc finger protein, putative 9 22.1 10 PF3D7_1426700 phosphoenolpyruvate carboxylase (PEPC) 17 22 20 PF3D7_1147500 farnesyltransferase beta subunit, putative 15 22 16 translation elongation factor EF-1, subunit PF3D7_1123400 alpha, putative 10 22 10 PF3D7_1325100 phosphoribosylpyrophosphate synthetase 6 22 3 nucleolar rRNA processing protein, PF3D7_0722600 putative 8 21.9 12 conserved Plasmodium protein, unknown PF3D7_1404900 function 3 21.9 4 PF3D7_0606700 coatomer alpha subunit, putative 23 21.7 25 PF3D7_0910100 Ran-binding protein, putative 20 21.7 18 PF3D7_1118300 insulinase, putative 23 21.6 26 PF3D7_0622800 leucine--tRNA ligase, putative 19 21.6 19 PF3D7_0219600 replication factor C subunit 1, putative 16 21.6 14 glutamate dehydrogenase, putative PF3D7_0802000 (GDH3) 19 21.5 22 conserved Plasmodium protein, unknown PF3D7_1010100 function 5 21.5 7 PF3D7_0618500 malate dehydrogenase (MDH) 3 21.4 2 conserved Plasmodium protein, unknown PF3D7_1147300 function 20 21.2 21 glutamine-dependent NAD(+) synthetase, PF3D7_0926700 putative (NADSYN) 13 21.2 12 conserved Plasmodium protein, unknown PF3D7_0407800 function 27 21.1 33 PF3D7_0517300 pre-mRNA-splicing factor (SR1) 6 21.1 7 serine/arginine-rich splicing factor 4 PF3D7_1022400 (SRSF4) 11 21 12 PF3D7_1124900 60S ribosomal protein L35, putative 3 21 4 conserved Plasmodium protein, unknown PF3D7_1036900 function 24 20.9 28 PF3D7_0823300 histone acetyltransferase GCN5 (GCN5) 23 20.9 27 beta subunit of coatomer complex, PF3D7_0905900 putative (SEC27) 15 20.8 15 PF3D7_0605100 RNA-binding protein, putative 12 20.8 10 PF3D7_0111800 conserved Plasmodium protein, unknown 11 20.8 11 170

function PF3D7_0923900 RNA-binding protein, putative 3 20.8 4 isocitrate dehydrogenase (NADP), PF3D7_1345700 mitochondrial precursor (IDH) 8 20.7 7 PF3D7_1142600 60S ribosomal protein L35ae, putative 4 20.7 4 26S proteasome regulatory subunit PF3D7_1368100 RPN11, putative (RPN11) 4 20.6 4 CCR4-NOT transcription complex subunit PF3D7_1103800 1, putative (NOT1) 43 20.5 48 PF3D7_1014300 conserved protein, unknown function 41 20.5 53 PF3D7_0320800 ATP-dependent RNA helicase DDX6 (DOZI) 7 20.3 7 secreted ookinete protein, putative PF3D7_0108700 (PSOP24) 13 20.2 14 conserved Plasmodium protein, unknown PF3D7_1249100 function 6 20.2 6 PF3D7_0602200 MYND finger protein, putative 5 20.2 4 ADP-ribosylation factor GTPase-activating PF3D7_1244600 protein, putative (ARFGAP) 4 20.2 4 phosphoinositide-binding protein, PF3D7_0704400 putative 11 20 12 PF3D7_1306600 vacuolar ATP synthase subunit h, putative 9 20 9 conserved Plasmodium protein, unknown PF3D7_0813000 function 7 20 6 conserved Plasmodium protein, unknown PF3D7_1465200 function 6 19.9 6 conserved Plasmodium protein, unknown PF3D7_1445700 function 5 19.9 4 26S protease regulatory subunit 8, PF3D7_1248900 putative (RPT6) 5 19.8 5 PF3D7_0913200 elongation factor 1-beta (EF-1beta) 4 19.6 4 mature parasite-infected erythrocyte surface antigen,erythrocyte membrane PF3D7_0500800 protein 2 (MESA) 19 19.5 25 conserved Plasmodium protein, unknown PF3D7_0105200 function 9 19.5 10 Fe-S cluster assembly protein DRE2, PF3D7_0824600 putative (DRE2) 4 19.5 3 PF3D7_1461900 valine--tRNA ligase, putative 13 19.4 16 gamma-glutamylcysteine synthetase PF3D7_0918900 (gammaGCS) 14 19.3 15 rRNA processing WD-repeat protein, PF3D7_1237600 putative 7 19.3 7 serine/threonine protein phosphatase 5 PF3D7_1355500 (PP5) 7 19.3 8 PF3D7_1429800 coatamer beta subunit, putative 19 19.2 21 regulator of nonsense transcripts, PF3D7_1005500 putative 18 19.2 21 NLI interacting factor-like phosphatase, PF3D7_0515900 putative (NIF2) 3 19.2 3 PF3D7_1241900 tetratricopeptide repeat protein, putative 10 19.1 11 171

erythrocyte membrane-associated PF3D7_0703500 antigen 29 19 31 PF3D7_0811300 CCR4-associated factor 1 (CAF1) 23 18.9 25 tetratricopeptide repeat family protein, PF3D7_1420200 putative 17 18.9 18 eukaryotic translation initiation factor PF3D7_1438000 eIF2A, putative 9 18.9 9 conserved Plasmodium protein, unknown PF3D7_1117900 function 19 18.8 20 splicing factor 3B subunit 3, putative PF3D7_1234800 (SF3B3) 16 18.8 15 H/ACA ribonucleoprotein complex subunit PF3D7_1309500 1, putative (GAR1) 3 18.7 3 histone S-adenosyl methyltransferase, PF3D7_1227800 putative 13 18.6 19 PF3D7_1228600 merozoite surface protein 9 (MSP9) 8 18.6 10 PF3D7_1037500 dynamin-like protein (DYN2) 7 18.6 7 conserved Plasmodium membrane PF3D7_1419400 protein, unknown function 28 18.5 30 PF3D7_1473200 DnaJ protein, putative 6 18.5 6 PF3D7_1426200 arginine methyltransferase 1 (PRMT1) 5 18.5 4 conserved Plasmodium protein, unknown PF3D7_0313000 function 4 18.5 4 mitochondrial-processing peptidase PF3D7_0933600 subunit beta, putative (MAS1) 7 18.4 7 conserved Plasmodium protein, unknown PF3D7_1140800 function 7 18.4 7 PF3D7_1416900 prefoldin subunit 2, putative 3 18.4 3 conserved Plasmodium protein, unknown PF3D7_1248700 function 24 18.3 25 conserved Plasmodium protein, unknown PF3D7_1227700 function 10 18.3 9 PF3D7_1334200 chaperone binding protein, putative 8 18.3 8 acid cluster protein 33 homologue, PF3D7_1441600 putative 8 18.3 8 replication factor C subunit 5, putative PF3D7_1111100 (RFC5) 4 18.3 5 3-oxo-5-alpha-steroid 4-dehydrogenase, PF3D7_1135900 putative 5 18.2 6 PF3D7_1331700 glutamine--tRNA ligase, putative 9 18.1 10 PF3D7_1330400.1;PF3 ER lumen protein retaining receptor 1, D7_1330400.2 putative 3 18 3 PF3D7_1435700.2;PF3 D7_1435700.1 ataxin-2 like protein, putative 9 17.9 11 conserved Plasmodium protein, unknown PF3D7_1141300 function 17 17.8 16 PF3D7_1238800 acyl-CoA synthetase (ACS11) 11 17.8 13 PF3D7_0823200 RNA-binding protein, putative 4 17.8 5 eukaryotic translation initation factor 4 PF3D7_1312900 gamma, putative (EIF4G) 15 17.7 15 172

PF3D7_1315900 exportin-1, putative 14 17.7 11 conserved Plasmodium protein, unknown PF3D7_0410800 function 19 17.6 21 PF3D7_1423700;REV_ conserved Plasmodium protein, unknown _PF3D7_1114900 function 21 17.5 25 PF3D7_0214100 protein transport protein SEC31 (SEC31) 15 17.5 16 DNA replication licensing factor MCM5, PF3D7_1211700 putative (MCM5) 9 17.4 8 conserved Plasmodium membrane PF3D7_0618000 protein, unknown function 7 17.4 7 PF3D7_1427900 conserved protein, unknown function 4 17.4 4 PF3D7_0818700 DNA helicase, putative 15 17.3 16 26S proteasome regulatory subunit RPN2, PF3D7_1466300 putative (RPN2) 13 17.2 15 calcium-dependent protein kinase 1 PF3D7_0217500 (CDPK1) 8 17.2 9 phenylalanyl-tRNA synthetase beta chain, PF3D7_1104000 putative 8 17.2 8 conserved Plasmodium protein, unknown PF3D7_0724700 function 44 17.1 42 PF3D7_0416400 histone acetyltransferase, putative (HAT1) 15 17.1 15 serine/threonine protein phosphatase 2B PF3D7_0802800 catalytic subunit A, putative (CNA) 5 17.1 4 FK506-binding protein (FKBP)-type PF3D7_1247400 peptidyl-prolyl isomerase (FKBP35) 4 17.1 4 S-adenosylmethionine decarboxylase/ornithine decarboxylase PF3D7_1033100 (AdoMetDC/ODC) 20 17 23 PF3D7_0922100 ubiquitin-like protein, putative 20 17 20 histone-arginine methyltransferase, PF3D7_0811500 putative (CARM1) 11 17 12 PF3D7_1132000 ubiquitin-like protein, putative 10 16.9 10 protein transport protein SEC61 subunit PF3D7_1346100 alpha (SEC61) 6 16.9 8 conserved Plasmodium protein, unknown PF3D7_0921900 function 5 16.9 6 PF3D7_0110500 bromodomain protein, putative 28 16.8 36 PF3D7_1333400 conserved protein, unknown function 3 16.8 3 PF3D7_1346300 DNA/RNA-binding protein Alba 2 (ALBA2) 4 16.6 3 conserved Plasmodium protein, unknown PF3D7_1328300 function 4 16.6 4 ubiquitin carboxyl-terminal hydrolase, PF3D7_0726500 putative 31 16.4 33 PF3D7_0728600 zinc finger, C3HC4 type, putative 27 16.4 29 AP endonuclease (DNA-[apurinic or PF3D7_0305600 apyrimidinic site] lyase), putative 8 16.4 8 26S proteasome regulatory subunit PF3D7_0807800 RPN10, putative (RPN10) 5 16.4 6 PF3D7_1104100 syntaxin, Qa-SNARE family (SYN13) 4 16.4 4 173

PF3D7_0704600 E3 ubiquitin-protein ligase (UT) 44 16.3 43 PF3D7_1104200 chromatin remodeling protein (SNF2L) 15 16.3 16 PF3D7_1455500 AP-1 complex subunit gamma, putative 11 16.3 10 PF3D7_1235600 serine hydroxymethyltransferase (SHMT) 6 16.3 5 PF3D7_0413500 phosphoglucomutase-2 (PGM2) 3 16.3 3 conserved Plasmodium protein, unknown PF3D7_1455700 function 11 16.2 12 Plasmodium exported protein (PHISTc), PF3D7_0936800 unknown function 4 16.2 4 conserved Plasmodium protein, unknown PF3D7_1244700 function 3 16.2 5 parasite-infected erythrocyte surface PF3D7_0310400 protein (PIESP1) 15 16 15 PF3D7_1433500 DNA topoisomerase II, putative 15 16 15 heat shock protein 101,chaperone protein PF3D7_1116800 ClpB2 (HSP101) 9 16 8 conserved Plasmodium protein, unknown PF3D7_0703600 function 3 16 3 PF3D7_1329100 myosin C (MyoC) 26 15.9 26 conserved Plasmodium protein, unknown PF3D7_0914100 function 16 15.9 17 PF3D7_1459000 ATP-dependent RNA helicase DBP5 (DBP5) 9 15.8 9 DEAD/DEAH box ATP-dependent RNA PF3D7_0521700 helicase, putative 9 15.8 8 pre-mRNA-splicing factor 38B, putative PF3D7_1407300 (PRP38B) 6 15.8 6 PF3D7_1445100 histidine--tRNA ligase, putative 14 15.7 14 nuclear protein localization protein 4, PF3D7_0507700 putative (NPL4) 6 15.6 5 conserved Plasmodium protein, unknown PF3D7_0630600 function 11 15.5 14 PF3D7_0528100 AP-1 complex subunit beta, putative 10 15.5 11 PF3D7_0803700 tubulin gamma chain (g-tub) 5 15.5 5 PF3D7_1008000 histone deacetylase 2 (HDA2) 28 15.4 29 PF3D7_0529400.1;PF3 conserved Plasmodium protein, unknown D7_0529400.2 function 21 15.4 24 PF3D7_1475600 bromodomain protein, putative 7 15.4 6 Plasmodium exported protein (PHISTa), PF3D7_0402000 unknown function 5 15.4 6 serine/threonine protein kinase, FIKK PF3D7_0424500 family (FIKK4.1) 8 15.3 7 pre-mRNA-splicing factor SLU7, putative PF3D7_0610100 (SLU7) 4 15.3 3 PF3D7_0420000 zinc finger protein, putative 40 15.2 58 PF3D7_0628200 protein kinase PK4 (PK4) 29 15.2 31 serine/threonine protein kinase, FIKK PF3D7_0805700 family (FIKK8) 17 15.2 19 double-strand break repair protein PF3D7_0107800 MRE11, putative (MRE11) 13 15.2 14 174

transcription factor with AP2 domain(s) PF3D7_0613800 (ApiAP2) 43 15.1 49 mRNA-decapping enzyme 2, putative PF3D7_1308900 (DCP2) 15 15.1 15 glucose-6-phosphate dehydrogenase-6- PF3D7_1453800 phosphogluconolactonase (G6PDH) 10 15.1 11 conserved Plasmodium protein, unknown PF3D7_1246300 function 9 15.1 10 PF3D7_1436600 cGMP-dependent protein kinase (PKG) 9 15.1 8 PF3D7_0811400 conserved protein, unknown function 6 15.1 6 PF3D7_1021800 schizont egress antigen-1 (SEA1) 23 15 25 transcription factor with AP2 domain(s) PF3D7_1007700 (ApiAP2) 22 15 22 ubiquitin carboxyl-terminal hydrolase, PF3D7_0527200 putative 7 15 9 translation initiation factor EIF-2b alpha PF3D7_0828500 subunit, putative 3 14.9 3 conserved Plasmodium protein, unknown PF3D7_1208900 function 15 14.8 15 inorganic anion exchanger, inorganic PF3D7_1471200 anion antiporter (SulP) 7 14.8 7 PF3D7_0501500 rhoptry-associated protein 3 (RAP3) 4 14.8 4 RNA-binding protein musashi, putative PF3D7_0916700 (HoMu) 5 14.7 6 PF3D7_0317800 proteasome regulatory protein, putative 3 14.7 3 conserved Plasmodium protein, unknown PF3D7_0604500 function 29 14.6 30 non-SERCA-type Ca2+ -transporting P- PF3D7_1211900 ATPase (ATP4) 11 14.6 11 transcription initiation factor TFIID PF3D7_0929000 subunit 7, putative (TAF7) 5 14.6 5 conserved Plasmodium protein, unknown PF3D7_1314100 function 9 14.5 10 rRNA biogenesis protein RRP5, putative PF3D7_1404500 (RRP5) 6 14.5 7 conserved Plasmodium protein, unknown PF3D7_1016200 function 4 14.5 6 PF3D7_0515000 RNA-binding protein, putative 8 14.4 9 dihydrofolate synthase/folylpolyglutamate synthase PF3D7_1324800 (DHFS-FPGS) 4 14.4 5 deubiquinating/deneddylating enzyme PF3D7_1117100 (UCH54) 4 14.4 5 U5 small nuclear ribonuclear protein, PF3D7_1003800 putative 11 14.3 10 DNA replication licensing factor MCM6 PF3D7_1355100 (MCM6) 10 14.3 12 root hair defective 3 GTP-binding protein PF3D7_1416100 (RHD3) homolog, putative 9 14.3 9 PF3D7_1033700 bromodomain protein, putative 7 14.3 6 175

conserved Plasmodium protein, unknown PF3D7_1128900 function 6 14.3 6 Plasmodium exported protein, unknown PF3D7_0831400 function 5 14.2 5 conserved Plasmodium protein, unknown PF3D7_1127900 function 4 14.2 5 conserved Plasmodium protein, unknown PF3D7_1442400 function 21 14.1 22 PF3D7_1419100 ATP-dependent RNA helicase, putative 12 14.1 11 conserved Plasmodium protein, unknown PF3D7_0813400 function 6 14.1 6 conserved Plasmodium protein, unknown PF3D7_0305200 function 6 14 6 conserved Plasmodium protein, unknown PF3D7_0706500 function 15 13.9 17 conserved Plasmodium protein, unknown PF3D7_0602600 function 12 13.9 15 PF3D7_1004400 RNA-binding protein, putative 10 13.9 10 conserved Plasmodium protein, unknown PF3D7_1238500 function 19 13.8 22 PF3D7_1412700 AAA family ATPase, putative 13 13.8 13 PF3D7_0321400 protein kinase, putative 5 13.8 5 PF3D7_0914700 transporter, putative 5 13.8 4 conserved Plasmodium protein, unknown PF3D7_0811600 function 12 13.7 10 ubiquitin conjugation factor E4 B, putative PF3D7_0826500 (UBE4B) 12 13.7 12 conserved Plasmodium protein, unknown PF3D7_1218900 function 9 13.7 11 PF3D7_1311400 AP-1 complex subunit mu, putative 3 13.7 4 pre-mRNA-processing-splicing factor 8, PF3D7_0405400 putative (PRPF8) 27 13.6 31 conserved Plasmodium protein, unknown PF3D7_0704800 function 10 13.6 10 PF3D7_0822600 protein transport protein SEC23 (SEC23) 8 13.6 8 protein transport protein Sec24A PF3D7_1361100 (SEC24A) 9 13.5 10 leucine carboxyl methyltransferase, PF3D7_1439700 putative 3 13.5 3 DNA mismatch repair protein MSH6, PF3D7_0505500 putative (MSH6) 14 13.4 13 conserved Plasmodium protein, unknown PF3D7_0507800 function 14 13.4 15 conserved Plasmodium protein, unknown PF3D7_0621100 function 12 13.4 9 PF3D7_0629400 RNA-binding protein, putative 7 13.3 6 conserved Plasmodium protein, unknown PF3D7_0723800 function 19 13.2 17 conserved Plasmodium protein, unknown PF3D7_0715200 function 17 13.2 19 176

conserved Plasmodium protein, unknown PF3D7_1419000 function 11 13.2 13 PF3D7_1342400 casein kinase II beta chain (CK2beta2) 4 13.2 4 ATP-specific succinyl-CoA synthetase beta PF3D7_1431600 subunit, putative 4 13.2 4 conserved Plasmodium protein, unknown PF3D7_0321100 function 17 13.1 18 PF3D7_1120500 tRNA nucleotidyltransferase, putative 7 13.1 8 conserved Plasmodium protein, unknown PF3D7_0524300 function 3 13.1 2 chromodomain-helicase-DNA-binding PF3D7_1023900 protein 1 homolog, putative (CHD1) 31 13 37 conserved Plasmodium protein, unknown PF3D7_1467600 function 19 13 28 conserved Plasmodium protein, unknown PF3D7_1225200 function 11 13 11 PF3D7_1304100 DNA ligase I (LigI) 8 13 11 transcription factor with AP2 domain(s) PF3D7_1466400 (ApiAP2) 8 13 9 PF3D7_1347100 DNA topoisomerase 3, putative (TOP3) 6 13 8 ubiquitin activating enzyme (E1) subunit PF3D7_1144500 Aos1, putative (AOS1) 4 13 4 PF3D7_1331300 signal peptidase 21 kDa subunit (SP21) 3 13 3 ubiquitin carboxyl-terminal hydrolase 1, PF3D7_0104300 putative (UBP1) 32 12.9 30 PF3D7_0903400 DEAD/DEAH box helicase, putative 22 12.9 23 P-loop containing nucleoside triphosphate PF3D7_1352700 hydrolase, putative 22 12.9 23 pre-mRNA-splicing factor ATP-dependent PF3D7_1231600 RNA helicase PRP2, putative (PRP2) 9 12.8 7 RNA binding protein Bruno, putative PF3D7_1409800 (HoBo) 5 12.8 6 PF3D7_0408500 flap endonuclease 1 (FEN1) 5 12.8 4 PF3D7_0304200 EH (Eps15 homology) protein (PAST1) 4 12.8 5 PF3D7_1466900 rhoptry protein, putative 12 12.7 11 cytoadherence linked asexual protein 8 PF3D7_0831600 (CLAG8) 8 12.7 8 PF3D7_1141800 phd finger protein, putative 4 12.7 4 PF3D7_1415000 uracil-DNA glycosylase (UDG) 3 12.7 3 PF3D7_1133800 RNA (uracil-5-)methyltransferase, putative 8 12.6 10 PF3D7_0306200 activator of Hsp90 ATPase (AHA1) 4 12.6 4 PF3D7_1307900 tripartite motif protein, putative 9 12.5 8 PF3D7_1343700 kelch protein K13 (K13) 5 12.5 5 conserved Plasmodium protein, unknown PF3D7_0218200 function 5 12.5 6 voltage-dependent anion-selective PF3D7_1432100 channel protein, putative 3 12.5 3 conserved Plasmodium protein, unknown PF3D7_1312800 function 23 12.4 25 177

PF3D7_1446200 M17 leucyl aminopeptidase (LAP) 6 12.4 6 PF3D7_1115200 SET domain protein, putative (SET7) 6 12.4 8 splicing factor 3B subunit 2, putative PF3D7_1461600 (SF3B2) 5 12.4 7 PF3D7_0629200 DnaJ protein, putative 4 12.4 4 PF3D7_0415000 arsenical pump-driving ATPase, putative 3 12.4 4 conserved Plasmodium membrane PF3D7_0903300 protein, unknown function 22 12.3 24 PF3D7_1357500 DNA helicase, putative 10 12.3 11 PF3D7_1251200 coronin 6 12.3 6 PF3D7_1005600 DnaJ protein, putative 6 12.3 6 PF3D7_0528800 nucleolar preribosomal GTPase, putative 5 12.3 6 rRNA processing and telomere PF3D7_0925200 maintaining methyltransferase, putative 4 12.3 4 phospholipid-transporting ATPase, PF3D7_1223400 putative 16 12.2 15 conserved Plasmodium protein, unknown PF3D7_1345800 function 10 12.2 8 mRNA binding Pumilio-homology domain PF3D7_0621300 protein, putative 9 12.2 9 conserved Plasmodium protein, unknown PF3D7_1419600 function 4 12.2 5 conserved Plasmodium protein, unknown PF3D7_1462300 function 10 12.1 10 DNA replication licensing factor MCM7, PF3D7_0705400 putative (MCM7) 7 12.1 6 DNA-directed RNA polymerase II subunit PF3D7_0318200 RPB1, putative (RPB1) 19 12 22 conserved Plasmodium protein, unknown PF3D7_0606000 function 9 12 9 conserved Plasmodium protein, unknown PF3D7_1126500 function 9 12 10 DNA repair helicase RAD3, putative PF3D7_0934100 (RAD3) 9 12 9 conserved Plasmodium protein, unknown PF3D7_0210900 function 3 12 3 conserved Plasmodium protein, unknown PF3D7_1332200 function 9 11.9 11 26S proteasome regulatory subunit RPN1, PF3D7_0205900 putative (RPN1) 7 11.9 8 N2,N2-dimethylguanosine tRNA PF3D7_1319300 methyltransferase, putative 7 11.9 7 transcription factor with AP2 domain(s), PF3D7_1449500 putative (ApiAP2) 7 11.9 7 PF3D7_1436000 glucose-6-phosphate isomerase (GPI) 6 11.9 7 conserved Plasmodium membrane PF3D7_0704100 protein, unknown function 31 11.8 30 conserved Plasmodium protein, unknown PF3D7_0420600 function 5 11.8 4 PF3D7_0804900 GTPase-activating protein, putative 4 11.8 4 178

conserved Plasmodium protein, unknown PF3D7_1142100 function 24 11.7 23 DNA-directed RNA polymerase II second PF3D7_0215700 largest subunit, putative 11 11.7 13 PF3D7_1126000 threonine--tRNA ligase (ThrRS) 10 11.7 10 26S proteasome regulatory subunit RPN7, PF3D7_1129200 putative (RPN7) 4 11.7 3 PF3D7_1330600 elongation factor Tu, putative 4 11.7 3 shewanella-like protein phosphatase 1, PF3D7_1469200 putative (SHLP1) 3 11.7 1 eukaryotic translation initiation factor 3 PF3D7_1007900 subunit 7, putative 6 11.6 6 structural maintenance of chromosome PF3D7_1130700 protein, putative 11 11.5 12 PF3D7_1212900 bromodomain protein, putative 9 11.5 6 conserved Plasmodium protein, unknown PF3D7_0922800 function 36 11.4 37 conserved Plasmodium protein, unknown PF3D7_0611600 function 4 11.4 3 PF3D7_0320500 nicotinamidase, putative (Nico) 3 11.4 3 PF3D7_1134800 coatomer delta subunit, putative 5 11.3 6 PF3D7_0314700 zinc finger protein, putative 11 11.2 12 PF3D7_0623100 coronin binding protein, putative 6 11.2 6 conserved Plasmodium membrane PF3D7_1137300 protein, unknown function 5 11.2 5 PF3D7_0920800 inosine-5-monophosphate dehydrogenase 4 11.2 4 PF3D7_1236100 clustered-asparagine-rich protein 4 11.2 4 conserved Plasmodium protein, unknown PF3D7_1202700 function 3 11.2 2 conserved Plasmodium protein, unknown PF3D7_1138000 function 22 11.1 26 conserved Plasmodium protein, unknown PF3D7_1002700 function 7 11.1 8 signal recognition particle receptor alpha PF3D7_1366600 subunit (SRPR-alpha) 5 11.1 5 PF3D7_1417200 NOT family protein, putative 35 11 35 PF3D7_0617100 AP-2 complex subunit alpha, putative 12 11 14 tetratricopeptide repeat family protein, PF3D7_1410000 putative 3 11 3 chromatin assembly factor 1 protein PF3D7_0110700 WD40 domain, putative 3 11 3 conserved Plasmodium protein, unknown PF3D7_0423300 function 7 10.9 15 Plasmodium exported protein (PHISTb), PF3D7_0401800 unknown function (PfD80) 7 10.9 8 conserved Plasmodium protein, unknown PF3D7_0111400 function (GEXP19) 6 10.9 6 26S proteasome regulatory subunit RPN9, PF3D7_1030500 putative (RPN9) 4 10.9 3 PF3D7_1235300 CCR4-NOT transcription complex subunit 14 10.8 18 179

4, putative (NOT4) conserved Plasmodium protein, unknown PF3D7_1237500 function 11 10.8 11 PF3D7_0720400 ferrodoxin reductase-like protein 5 10.7 5 glideosome-associated protein 40, PF3D7_0515700 putative (GAP40) 4 10.7 3 ribose-phosphate pyrophosphokinase, PF3D7_1327800 putative 3 10.7 2 PF3D7_1110400 asparagine-rich antigen 11 10.6 12 PF3D7_1139100 RNA-binding protein, putative 15 10.5 16 ATP-dependent protease ATPase subunit PF3D7_0907400 ClpY (ClpY) 5 10.5 5 PF3D7_0925700 histone deacetylase (HDAC1) 3 10.5 3 multidrug resistance-associated protein 1 PF3D7_0112200 (MRP1) 12 10.4 13 conserved Plasmodium protein, unknown PF3D7_1343300 function 6 10.4 5 PF3D7_0824400 nucleoside transporter 2 (NT2) 5 10.4 6 PF3D7_1442300 tRNA binding protein, putative 4 10.4 5 eukaryotic translation initiation factor 5, PF3D7_1206700 putative 4 10.4 4 conserved Plasmodium protein, unknown PF3D7_0619700 function 5 10.3 5 vacuolar proton translocating ATPase PF3D7_0806800 subunit A, putative 6 10.2 7 PF3D7_0629800 cullin-like protein, putative 6 10.2 7 Plasmodium exported protein (PHISTb), PF3D7_0532300 unknown function 3 10.2 3 prolyl 4-hydroxylase subunit alpha, PF3D7_0829400 putative 3 10.2 3 origin recognition complex subunit 1 PF3D7_1203000 (ORC1) 8 10.1 8 PF3D7_0931000 elongation factor Tu, putative 5 10.1 5 phosphopantetheine adenylyltransferase, PF3D7_0704700 putative (PPAT) 11 10 8 PF3D7_1239000 HD superfamily phosphohydrolase protein 6 10 7 PF3D7_1131100 serpentine receptor, putative (SR1) 4 10 4 PF3D7_1473100 GTPase-activating protein, putative 4 10 4 PF3D7_1103600 actin-like protein, putative (ALP2a) 4 10 4 U4/U6 small nuclear ribonucleoprotein PF3D7_1309300 PRP3, putative (PRPF3) 3 10 3 pre-mRNA-processing factor 6, putative PF3D7_1110200 (PRPF6) 7 9.9 7 transcription or splicing factor-like PF3D7_0623600 protein, putative 4 9.9 4 conserved Plasmodium protein, unknown PF3D7_1236900 function 3 9.9 3 PF3D7_0613700 syntaxin binding protein, putative 5 9.8 3 PF3D7_1247700 conserved Plasmodium protein, unknown 3 9.8 3 180

function conserved Plasmodium protein, unknown PF3D7_1234100 function 27 9.7 30 conserved Plasmodium protein, unknown PF3D7_1138800 function 9 9.7 10 transcription factor with AP2 domain(s) PF3D7_0516800 (ApiAP2) 14 9.6 17 conserved Plasmodium protein, unknown PF3D7_0820900 function 10 9.6 8 conserved Plasmodium protein, unknown PF3D7_0824900 function 7 9.6 5 conserved Plasmodium protein, unknown PF3D7_0912400 function 3 9.6 3 PF3D7_0926100 protein kinase, putative 28 9.5 40 conserved Plasmodium protein, unknown PF3D7_0707200 function 15 9.5 15 PF3D7_0613900 myosin E, putative (myoE) 15 9.4 16 conserved Plasmodium protein, unknown PF3D7_0927600 function 5 9.4 5 DEAD/DEAH box ATP-dependent RNA PF3D7_0218400 helicase, putative 4 9.4 4 PF3D7_0112000 TatD-like deoxyribonuclease, putative 3 9.4 3 conserved Plasmodium protein, unknown PF3D7_0607700 function 14 9.3 13 PF3D7_1235500 mRNA methyltransferase, putative 5 9.3 5 PF3D7_1115400 cysteine proteinase falcipain 3 (FP3) 4 9.3 4 conserved Plasmodium membrane PF3D7_0704300 protein, unknown function 10 9.2 10 PF3D7_1218300 AP-3 complex subunit mu, putative 3 9.2 4 PF3D7_1465300 tRNA 3-trailer sequence RNase, putative 7 9.1 8 U4/U6 small nuclear ribonucleoprotein PF3D7_1343900 PRP4, putative (PRPF4) 4 9.1 5 guanine nucleotide-exchange factor PF3D7_1116400 SEC12 (SEC12) 3 9.1 3 protein arginine N-methyltransferase 5, PF3D7_1361000 putative (PRMT5) 4 9 3 pre-mRNA-splicing helicase BRR2, PF3D7_0422500 putative (BRR2) 16 8.9 17 conserved Plasmodium protein, unknown PF3D7_0418300 function 6 8.8 6 conserved Plasmodium protein, unknown PF3D7_0609700 function 12 8.7 13 transcription factor with AP2 PF3D7_0604100 domain(s),SPE2-interacting protein (SIP2) 12 8.7 12 cytoadherence linked asexual protein 2 PF3D7_0220800 (CLAG2) 6 8.7 4 conserved Plasmodium membrane PF3D7_0721000 protein, unknown function 20 8.6 20 phosphatidylinositol 3- and 4-kinase, PF3D7_0311300 putative 6 8.6 5 181

conserved Plasmodium protein, unknown PF3D7_1228800 function 20 8.5 20 PF3D7_1121700 protein GCN20 (GCN20) 5 8.5 6 conserved Plasmodium protein, unknown PF3D7_0105800 function 5 8.5 4 PF3D7_0709000 chloroquine resistance transporter (CRT) 4 8.5 4 polyadenylate-binding protein-interacting PF3D7_1107300 protein 1, putative (PAIP1) 18 8.4 17 conserved Plasmodium protein, unknown PF3D7_0410900 function 5 8.4 6 conserved Plasmodium protein, unknown PF3D7_1111700 function 5 8.4 5 conserved Plasmodium protein, unknown PF3D7_1315300 function 3 8.4 3 PF3D7_1438400 metacaspase-like protein (MCA2) 12 8.3 14 conserved Plasmodium protein, unknown PF3D7_0408100 function 5 8.3 7 conserved Plasmodium protein, unknown PF3D7_1206800 function 5 8.3 3 PF3D7_0217100 ATP synthase F1, alpha subunit 5 8.2 5 conserved Plasmodium protein, unknown PF3D7_1203300 function 5 8.2 5 serine/threonine protein kinase, FIKK PF3D7_1016400 family (FIKK10.1) 3 8.2 2 splicing factor 3A subunit 3, putative PF3D7_0924700 (SF3A3) 4 8.1 4 PF3D7_1012500 phosphoglucomutase, putative 3 8.1 3 snoRNA-associated small subunit rRNA PF3D7_1217200 processing protein, putative 7 8 8 PF3D7_1216900 DNA-binding chaperone, putative 6 8 6 PF3D7_0317500 kinesin-5 (EG5) 10 7.9 9 PF3D7_1429900 ATP-dependent DNA helicase, putative 9 7.9 5 vacuolar protein sorting-associated PF3D7_1110500 protein 35, putative (VPS35) 7 7.9 8 conserved Plasmodium protein, unknown PF3D7_1015400 function 5 7.9 5 DEAD/DEAH box ATP-dependent RNA PF3D7_1251500 helicase, putative 4 7.9 4 PF3D7_0204700 hexose transporter (HT) 3 7.9 3 PF3D7_0206000 DNA repair endonuclease, putative 10 7.8 12 conserved Plasmodium protein, unknown PF3D7_1133200 function 10 7.8 11 chromosome segregation protein, PF3D7_1318400 putative 8 7.8 7 vacuolar protein sorting-associated PF3D7_1239900 protein 16, putative (VPS16) 5 7.8 5 conserved Plasmodium protein, unknown PF3D7_0214400 function 4 7.8 4 PF3D7_1247500 serine/threonine protein kinase, putative 4 7.8 4 PF3D7_1130000 phosphoacetylglucosamine mutase, 4 7.8 3 182

putative (PAGM) PF3D7_1437400 pantothenate kinase, putative 3 7.8 4 conserved Plasmodium protein, unknown PF3D7_1321100 function 19 7.7 20 PF3D7_0523400 DnaJ protein, putative 4 7.7 4 regulator of chromosome condensation, PF3D7_0919900 putative 15 7.6 16 conserved Plasmodium protein, unknown PF3D7_1459600 function 4 7.6 4 conserved Plasmodium protein, unknown PF3D7_1245500 function 3 7.6 3 PF3D7_0317200 cdc2-related protein kinase 4 (CRK4) 8 7.5 6 N-ethylmaleimide-sensitive fusion protein PF3D7_0303000 (NSF) 4 7.5 4 transcription factor with AP2 domain(s) PF3D7_1107800 (ApiAP2) 9 7.3 9 conserved Plasmodium protein, unknown PF3D7_1352400 function 8 7.3 8 conserved Plasmodium protein, unknown PF3D7_1305000 function 7 7.3 7 nucleoporin NUP116/NSP116, putative PF3D7_1473700 (NUP116) 6 7.3 5 PF3D7_1017000 DNA polymerase delta catalytic subunit 5 7.3 5 PF3D7_0934700 UBX domain, putative 4 7.3 4 conserved Plasmodium protein, unknown PF3D7_1207000 function 16 7.2 14 conserved Plasmodium protein, unknown PF3D7_0903500 function 6 7.2 7 DNA repair endonuclease, putative PF3D7_1368800 (ERCC4) 8 7.1 8 multidrug resistance protein 2 (heavy PF3D7_1447900 metal transport family) (MDR2) 5 7.1 4 PF3D7_1230700 protein transport protein SEC13 (SEC13) 4 7.1 5 conserved Plasmodium protein, unknown PF3D7_0817600 function 4 7.1 3 myb2 transcription factor, putative PF3D7_1033600 (Myb2) 4 7.1 4 PF3D7_0511500 RNA pseudouridylate synthase, putative 48 7 51 PF3D7_1128000.1;PF3 conserved Plasmodium protein, unknown D7_1128000.2 function 9 7 9 conserved Plasmodium protein, unknown PF3D7_1409600 function 3 7 3 PF3D7_0417800 cdc2-related protein kinase 1 (CRK1) 3 7 3 PF3D7_0102500 erythrocyte binding antigen-181 (EBA181) 8 6.9 7 conserved Plasmodium protein, unknown PF3D7_1303400 function 5 6.9 5 PF3D7_1463300 DNA polymerase alpha subunit, putative 3 6.9 3 conserved Plasmodium protein, unknown PF3D7_0508900 function 14 6.8 12 PF3D7_1022000 RNA-binding protein, putative 7 6.8 7 183

polypyrimidine tract binding protein, PF3D7_0606500 putative 3 6.8 3 PF3D7_0629700 SET domain protein, putative (SET1) 33 6.7 34 PF3D7_0904100 AP-4 complex subunit epsilon, putative 6 6.7 7 PF3D7_0910400 selenide water dikinase, putative 4 6.7 5 conserved Plasmodium protein, unknown PF3D7_0725300 function 3 6.7 2 conserved Plasmodium protein, unknown PF3D7_1107200 function 3 6.7 2 conserved Plasmodium protein, unknown PF3D7_0729100 function 11 6.6 12 PF3D7_1134500 alpha/beta hydrolase, putative 10 6.6 9 conserved Plasmodium protein, unknown PF3D7_0822900 function 6 6.6 7 replication protein A1, large subunit PF3D7_0409600 (RPA1) 5 6.6 6 PF3D7_1468400 zinc finger protein, putative (D13) 4 6.6 4 U4/U6.U5 tri-snRNP-associated protein 2, PF3D7_1317000 putative (USP39) 3 6.6 3 conserved Plasmodium protein, unknown PF3D7_1317600 function 3 6.6 3 N-alpha-acetyltransferase 15, NatA PF3D7_1244100 auxiliary subunit, putative 7 6.5 6 PF3D7_1245100 kinesin-13, putative (KLP8) 6 6.5 6 vacuolar protein sorting-associated PF3D7_0935200 protein 33, putative (VPS33) 5 6.5 5 PF3D7_0919800 TLD domain-containing protein 5 6.5 5 conserved Plasmodium protein, unknown PF3D7_0606600 function 13 6.4 11 conserved Plasmodium protein, unknown PF3D7_1369500 function 5 6.4 4 PF3D7_1437700 succinyl-CoA ligase, putative 5 6.4 2 conserved Plasmodium protein, unknown PF3D7_1316900 function 4 6.4 4 conserved Plasmodium membrane PF3D7_1308000 protein, unknown function 4 6.4 3 conserved Plasmodium protein, unknown PF3D7_1008100 function 16 6.3 13 calponin homology domain-containing PF3D7_1447800 protein, putative 9 6.3 11 conserved Plasmodium protein, unknown PF3D7_1020200 function 4 6.3 4 conserved Plasmodium protein, unknown PF3D7_1310800 function 6 6.2 5 PF3D7_0602100 ATP-dependent RNA helicase, putative 5 6.2 4 conserved Plasmodium protein, unknown PF3D7_0824200 function 5 6.2 5 PF3D7_1129900 transporter, putative 3 6.2 4 conserved Plasmodium protein, unknown PF3D7_0721200 function 3 6.2 3 184

chromosome condensation protein, PF3D7_0509100 putative 8 6.1 5 PF3D7_1321700 splicing factor 1 (SF1) 3 6.1 4 conserved Plasmodium protein, unknown PF3D7_1455300 function 3 6.1 2 PF3D7_0705500 inositol-phosphate phosphatase, putative 11 6 12 conserved Plasmodium protein, unknown PF3D7_1119900 function 7 6 8 conserved Plasmodium protein, unknown PF3D7_0418100 function 5 6 4 conserved Plasmodium protein, unknown PF3D7_0714200 function 5 6 4 conserved Plasmodium membrane PF3D7_0824800 protein, unknown function 6 5.9 4 E3 SUMO-protein ligase PIAS, putative PF3D7_1360700 (PIAS) 4 5.9 2 conserved Plasmodium protein, unknown PF3D7_1443400 function 3 5.9 3 dual specificity protein phosphatase PF3D7_0309000 (YVH1) 3 5.9 3 carbon catabolite repressor protein 4, PF3D7_0519500 putative (CCR4) 12 5.8 11 transcription factor with AP2 domain(s) PF3D7_1456000 (ApiAP2) 5 5.8 5 conserved Plasmodium protein, unknown PF3D7_1343800 function 30 5.7 24 PF3D7_0704200 tRNA m5C-methyltransferase, putative 5 5.7 5 SWI/SNF-related matrix-associated actin- PF3D7_0611400 dependent regulator of chromatin 3 5.7 3 conserved Plasmodium protein, unknown PF3D7_0207100 function 7 5.6 6 conserved Plasmodium protein, unknown PF3D7_1019600 function 4 5.6 3 PF3D7_0506800 transcription factor 25, putative (TCF25) 3 5.6 4 conserved Plasmodium protein, unknown PF3D7_1366900 function 3 5.5 4 PF3D7_0202600.2;PF3 conserved Plasmodium protein, unknown D7_0202600.1 function 3 5.5 4 PF3D7_1014600 transcriptional coactivator ADA2 (ADA2) 9 5.4 11 serine/threonine protein phosphatase 8, PF3D7_1018200 putative (PPP8) 8 5.4 8 PF3D7_0506900 rhomboid protease ROM4 (ROM4) 3 5.4 3 conserved Plasmodium protein, unknown PF3D7_0613600 function 3 5.4 3 conserved Plasmodium protein, unknown PF3D7_0913600 function 4 5.3 3 ubiquitin carboxyl-terminal hydrolase 2, PF3D7_0516700 putative 4 5.3 3 PF3D7_0810300 protein phosphatase, putative 3 5.3 3 PF3D7_1406200 conserved Plasmodium protein, unknown 11 5.2 11 185

function PF3D7_0411900 DNA polymerase alpha 7 5.2 7 60S ribosomal export protein NMD3, PF3D7_0729300 putative (NMD3) 4 5.2 4 rRNA processing WD-repeat protein, PF3D7_0802300 putative 4 5.2 3 large subunit rRNA processing protein, PF3D7_1405800 putative 3 5.2 3 asparagine and aspartate rich protein 1 PF3D7_1233600 (AARP1) 16 5.1 15 conserved Plasmodium membrane PF3D7_0212400 protein, unknown function 14 5.1 15 vacuolar protein sorting-associated PF3D7_1423800 protein 3, putative (VPS3) 6 5.1 4 conserved Plasmodium membrane PF3D7_0305300 protein, unknown function 3 5.1 4 splicing factor 3B subunit 1, putative PF3D7_0308900 (SF3B1) 3 5.1 3 PF3D7_0811200 conserved protein, unknown function 3 5.1 3 conserved Plasmodium protein, unknown PF3D7_1339700 function 7 5 6 conserved Plasmodium protein, unknown PF3D7_1213900 function 6 5 5 PF3D7_1344100 krox-like protein, putative 3 5 5

MTIPB-BIRA* sample Sequence MS/MS Protein IDs Product Description Peptides coverage] count PF3D7_0922500 phosphoglycerate kinase (PGK) 42 87.5 59 glyceraldehyde-3-phosphate PF3D7_1462800 dehydrogenase (GAPDH) 22 80.7 47 PF3D7_0626800 pyruvate kinase (PyrK) 34 80.4 73 PF3D7_1357100;PF3D 7_1357000 elongation factor 1-alpha 35 78.8 147 PF3D7_0306800 T-complex protein beta subunit, putative 35 77.9 58 GTP-binding nuclear protein RAN/TC4 PF3D7_1117700 (RAN) 15 77.1 37 PF3D7_1026800 40S ribosomal protein S2 (RPS2) 14 73 15 conserved Plasmodium protein, unknown PF3D7_1118700 function 64 72.4 51 PF3D7_0516200 40S ribosomal protein S11 9 72.2 9 PF3D7_0708400 heat shock protein 90 (HSP90) 72 72.1 586 PF3D7_1246200 actin I (ACT1) 22 71.5 43 phosphoethanolamine N- PF3D7_1343000 methyltransferase (PMT) 17 70.7 41 PF3D7_1015900 enolase (ENO) 30 70.6 28 PF3D7_1357800 TCP-1/cpn60 chaperonin family, putative 29 70.1 27 PF3D7_1124600 ethanolamine kinase (EK) 24 70 38 PF3D7_1008700 tubulin beta chain 22 69.9 32 186

PF3D7_0416800 small GTP-binding protein sar1 (SAR1) 9 69.8 17 PF3D7_0608800 ornithine aminotransferase (OAT) 27 69.1 39 hypoxanthine-guanine PF3D7_1012400 phosphoribosyltransferase (HGPRT) 15 68.8 18 ATP-dependent RNA helicase UAP56 PF3D7_0209800 (UAP56) 24 68.1 40 PF3D7_1130200 60S ribosomal protein P0 (PfP0) 14 68 15 PF3D7_1426000 60S ribosomal protein L21 (RPL21) 13 67.7 20 PF3D7_1451100 elongation factor 2 45 67.5 85 PF3D7_1444800 fructose-bisphosphate aldolase (FBPA) 22 67.5 35 PF3D7_0322900 40S ribosomal protein S3A, putative 26 67.2 33 PF3D7_0722400 GTP-binding protein, putative 18 67.2 29 PF3D7_1016300;PF3D 7_0937000 glycophorin binding protein (GBP) 11 66.4 67 40S ribosomal protein S11, putative PF3D7_0317600 (RPS11) 15 65.8 18 PF3D7_0624000 hexokinase (HK) 34 65.5 47 PF3D7_1331800 60S ribosomal protein L23, putative 9 65.5 14 PF3D7_0818900;PF3D 7_0831700 heat shock protein 70 (HSP70) 55 65.1 179 PF3D7_1105400 40S ribosomal protein S4, putative 20 64.8 34 PF3D7_0316800 40S ribosomal protein S15A, putative 9 64.6 13 PF3D7_0922200 S-adenosylmethionine synthetase (SAMS) 22 64.2 41 PF3D7_1468700 eukaryotic initiation factor 4A (eIF4A) 26 64.1 32 PF3D7_0818200 14-3-3 protein (14-3-3I) 15 63.7 29 PF3D7_1460700 60S ribosomal protein L27 (RPL27) 12 63.7 14 PF3D7_1302800 40S ribosomal protein S7, putative 17 62.9 22 haloacid dehalogenase-like hydrolase PF3D7_1033400 (HAD1) 10 62.8 13 PF3D7_1224000 GTP cyclohydrolase I (GCH1) 27 62.7 45 high molecular weight rhoptry protein 2 PF3D7_0929400 (RhopH2) 86 62.6 146 PF3D7_0524000 karyopherin beta (KASbeta) 56 62.1 91 PF3D7_1105000 histone H4 (H4) 14 62.1 31 PF3D7_1324900 L-lactate dehydrogenase (LDH) 12 61.7 14 PF3D7_1108400 casein kinase 2, alpha subunit (CK2alpha) 17 61.5 26 PF3D7_0422400 40S ribosomal protein S19 (RPS19) 12 61.2 15 PF3D7_1437900 HSP40, subfamily A, putative 20 60.4 21 PF3D7_1020900 ADP-ribosylation factor (ARF1) 9 60.2 15 PF3D7_1421200 40S ribosomal protein S25 (RPS25) 7 60 6 PF3D7_0406100 vacuolar ATP synthase subunit b 21 59.5 29 PF3D7_1302100 gamete antigen 27/25 (Pfg27) 11 59.4 13 PF3D7_1019400 60S ribosomal protein L30e, putative 6 59.3 7 PF3D7_1424100 60S ribosomal protein L5, putative 20 59.2 28 PF3D7_1338200 60S ribosomal protein L6-2, putative 13 58.8 19 PF3D7_0721600 40S ribosomal protein S5, putative 12 58.5 16 187

PF3D7_0507100 60S ribosomal protein L4 (RPL4) 28 58.4 63 PF3D7_1027800 60S ribosomal protein L3 (RPL3) 25 58 48 box C/D snoRNP rRNA 2-O-methylation PF3D7_1118500 factor, putative 31 57.9 46 conserved Plasmodium protein, unknown PF3D7_0513200 function 173 57.7 138 PF3D7_0517000 60S ribosomal protein L12, putative 7 57.6 4 PF3D7_0617800 histone H2A (H2A) 5 57.6 16 heat shock protein DNAJ homologue Pfj4 PF3D7_1211400 (PfJ4) 10 57.4 10 PF3D7_1323100 60S ribosomal protein L6, putative 8 57.4 11 PF3D7_1011800 PRE-binding protein (PREBP) 73 57.2 127 PF3D7_1242700 40S ribosomal protein S17, putative 8 56.9 9 PF3D7_1136300 tudor staphylococcal nuclease (TSN) 62 56.7 64 PF3D7_1465900 40S ribosomal protein S3 17 56.6 28 PF3D7_1407100 fibrillarin, putative (NOP1) 14 56.6 16 PF3D7_1446600 centrin-2 (CEN2) 4 56.5 11 PF3D7_1006200 DNA/RNA-binding protein Alba 3 (ALBA3) 4 56.1 3 PF3D7_0510500 topoisomerase I (TopoI) 47 55.9 81 PF3D7_1311900 vacuolar ATP synthase subunit a (vapA) 20 55.8 24 PF3D7_0807300 ras-related protein Rab-18 (RAB18) 8 55.7 10 PF3D7_1202900 high mobility group protein B1 (HMGB1) 5 55.7 7 PF3D7_0615400 ribonuclease, putative 167 55.4 224 PF3D7_1130100 60S ribosomal protein L38 (RPL38) 8 55.2 6 PF3D7_1126200 40S ribosomal protein S18, putative 8 55.1 6 T-complex protein 1, gamma subunit, PF3D7_1229500 putative 26 54.4 35 PF3D7_0610400 histone H3 (H3) 9 54.4 16 PF3D7_1414300 60S ribosomal protein L10, putative 15 54.3 36 PF3D7_1341200 60S ribosomal protein L18, putative 14 54.3 20 PF3D7_1410400 rhoptry-associated protein 1 (RAP1) 34 54.2 26 PF3D7_0308200 TCP-1/cpn60 chaperonin family, putative 19 54.2 30 PF3D7_0516900 60S ribosomal protein L2 (RPL2) 12 54.2 18 PF3D7_0503400 actin-depolymerizing factor 1 (ADF1) 5 54.1 9 PF3D7_1438900 thioredoxin peroxidase 1 (Trx-Px1) 7 53.8 10 PF3D7_0501600 rhoptry-associated protein 2 (RAP2) 18 53.5 16 PF3D7_1347500 DNA/RNA-binding protein Alba 4 (ALBA4) 17 53.5 23 PF3D7_0214000 T-complex protein 1, putative 24 53.3 22 6-phosphogluconate dehydrogenase, PF3D7_1454700 decarboxylating, putative 17 52.8 16 PF3D7_0813900 40S ribosomal protein S16, putative 10 52.8 14 PF3D7_0525100 acyl-CoA synthetase (ACS10) 23 52.7 18 PF3D7_1034900 methionine--tRNA ligase, putative 43 52.5 48 PF3D7_1408600 40S ribosomal protein S8e, putative 11 52.3 19 eukaryotic translation initiation factor 2 PF3D7_1010600 beta subunit, putative 10 52.3 12 188

conserved Plasmodium protein, unknown PF3D7_1457300 function 23 51.5 28 PF3D7_0413700 lysine decarboxylase-like protein, putative 11 51.5 7 PF3D7_0812400 karyopherin alpha (KARalpha) 19 51.4 22 PF3D7_0915400 6-phosphofructokinase (PFK9) 53 51.2 75 PF3D7_1136500.1;PF 3D7_1136500.2 casein kinase 1 (CK1) 19 51.1 22 PF3D7_1317800 40S ribosomal protein S19 (RPS19) 7 51 17 PF3D7_0608700 chaperone, putative 21 50.8 25 phosphoglycerate mutase, putative PF3D7_1120100 (PGM1) 12 50.8 18 PF3D7_0617900 histone H3 variant, putative (H3.3) 4 50.7 7 signal recognition particle subunit SRP54 PF3D7_1450100 (SRP54) 19 50.4 19 PF3D7_1447000 40S ribosomal protein S5 14 50.4 23 PF3D7_0823800 DnaJ protein, putative 28 50.2 34 PF3D7_1336400 RanBPM and CLTH-like protein, putative 8 50 8 PF3D7_0827900 protein disulfide isomerase (PDI8) 13 49.7 12 PF3D7_1358800 40S ribosomal protein S15 (RPS15) 10 49.7 9 conserved Plasmodium protein, unknown PF3D7_1466800 function 40 49.1 46 PF3D7_1349200 glutamate--tRNA ligase, putative 37 48.9 34 PF3D7_1308300 40S ribosomal protein S27 (RPS27) 5 48.8 5 PF3D7_0826700 receptor for activated c kinase (RACK) 10 48.6 18 26S protease regulatory subunit 7, putative PF3D7_1311500 (RPT1) 13 47.9 23 cytoadherence linked asexual protein 3.1 PF3D7_0302500 (CLAG3.1) 62 47.6 105 PF3D7_0814000 60S ribosomal protein L13-2, putative 11 47.4 18 PF3D7_1424400 60S ribosomal protein L7-3, putative 20 47.3 28 PF3D7_0917900 heat shock protein 70 (HSP70-2) 31 47.2 42 PF3D7_0307200 60S ribosomal protein L7, putative 14 47.1 23 PF3D7_0627500 protein DJ-1 (DJ1) 6 47.1 4 PF3D7_1222300 endoplasmin, putative (GRP94) 31 46.9 30 PF3D7_0706400 60S ribosomal protein L37 (RPL37) 5 46.7 6 PF3D7_0520000 40S ribosomal protein S9, putative 10 46.6 8 PF3D7_0606900 glutaredoxin-like protein (GLP2) 6 46.6 6 PF3D7_1340300 nucleolar complex protein 2, putative 40 46.5 67 eukaryotic translation initiation factor 3 PF3D7_0612100 subunit L, putative 21 46.5 26 signal recognition particle subunit SRP72, PF3D7_1136400 putative (SRP72) 33 46.3 35 cAMP-dependent protein kinase catalytic PF3D7_0934800 subunit (PKAc) 17 46.2 22 PF3D7_1105100 histone H2B (H2B) 8 46.2 34 PF3D7_0320900 histone H2A variant, putative (H2A.Z) 6 46.2 6 PF3D7_0904800 replication protein A1, small fragment 18 46.1 20 189

(RPA1) PF3D7_1238100 calcyclin binding protein, putative 8 46.1 11 PF3D7_0512600 ras-related protein Rab-1B (RAB1b) 7 46 7 PF3D7_1416900 prefoldin subunit 2, putative 6 45.6 3 PF3D7_1472200 histone deacetylase, putative (HDA1) 107 45.5 139 T-complex protein 1 epsilon subunit, PF3D7_0320300 putative 23 45.4 28 26S proteasome AAA-ATPase subunit PF3D7_0413600 RPT3, putative 11 45.4 11 PF3D7_0618300 60S ribosomal protein L27a, putative 8 45.3 14 PF3D7_0511800 inositol-3-phosphate synthase (INO1) 22 45.2 16 PF3D7_1358700 HVA22-like protein, putative 6 45.1 4 PF3D7_1025000 formin 2, putative 40 44.8 70 PF3D7_1338300 elongation factor 1-gamma, putative 16 44.3 25 PF3D7_1201500 XPA binding protein 1, putative 14 44.1 9 eukaryotic translation initiation factor 2 PF3D7_1410600 gamma subunit, putative 14 43.9 18 PF3D7_1342000 40S ribosomal protein S6 23 43.8 76 26S protease regulatory subunit 10B, PF3D7_1306400 putative (RPT4) 12 43.8 15 proliferation-associated protein 2g4, PF3D7_1428300 putative 17 43.5 18 ADP/ATP transporter on adenylate PF3D7_1037300 translocase (ADT) 12 43.5 16 eukaryotic translation initiation factor 3 PF3D7_0918300 subunit 5, putative 8 43.4 8 PF3D7_0814200 DNA/RNA-binding protein Alba 1 (ALBA1) 10 43.1 17 PF3D7_1006800 RNA-binding protein, putative 11 42.7 17 PF3D7_0716600 cysteine desulfurase (SufS) 17 42.5 23 PF3D7_1420600 pantothenate kinase, putative (PANK) 16 42.4 19 PF3D7_0709700;PF3D 7_0102400 lysophospholipase, putative 9 42.4 16 glutathione peroxidase-like thioredoxin PF3D7_1212000 peroxidase (TPx(Gl)) 7 42.4 11 PF3D7_0708800 heat shock protein 110 (HSP110c) 24 42.2 36 PF3D7_0501500 rhoptry-associated protein 3 (RAP3) 15 42.2 4 PF3D7_1346300 DNA/RNA-binding protein Alba 2 (ALBA2) 8 42.2 3 PF3D7_0719600 60S ribosomal protein L11a, putative 7 42.2 8 ATP-dependent RNA helicase DDX5, PF3D7_1445900 putative (DDX5) 17 42.1 19 PF3D7_1323400 60S ribosomal protein L23 (RPL23) 13 42.1 22 lysine-rich membrane-associated PHISTb PF3D7_0532400 protein (LyMP) 18 41.9 20 PF3D7_1008800 nucleolar protein 5, putative (NOP5) 16 41.8 23 PF3D7_1332900 isoleucine--tRNA ligase, putative 41 41.7 46 H/ACA ribonucleoprotein complex subunit PF3D7_1417500 4, putative (CBF5) 11 41.6 14 PF3D7_0520900 S-adenosyl-L-homocysteine hydrolase 20 41.3 39 190

(SAHH) PF3D7_1431700 60S ribosomal protein L14, putative 8 41.2 5 inner membrane complex sub- PF3D7_1460600 compartment protein 3, putative (ISP3) 4 41.2 5 PF3D7_0110400 DNA-directed RNA polymerase 2, putative 11 41 28 PF3D7_0315100 translation initiation factor 4E (eIF4E) 7 41 6 conserved Plasmodium membrane protein, PF3D7_1237700 unknown function 6 41 8 PF3D7_0802200 1-cys peroxiredoxin (1-CysPxn) 5 40.9 9 PF3D7_1003500 40S ribosomal protein S20e, putative 6 40.7 10 conserved Plasmodium protein, unknown PF3D7_1241100 function 12 40.6 7 PF3D7_1426100 basic transcription factor 3b, putative 7 40.4 8 PF3D7_0821700 60S ribosomal protein L22, putative 7 40.3 11 PF3D7_0217800 40S ribosomal protein S26 (RPS26) 5 40.2 4 conserved Plasmodium protein, unknown PF3D7_1002900 function 3 40.2 5 PF3D7_1132200 TCP-1/cpn60 chaperonin family, putative 16 40.1 10 PF3D7_1341300 60S ribosomal protein L18-2, putative 7 40.1 8 eukaryotic translation initiation factor, PF3D7_0517700 putative 20 39.9 11 PF3D7_1231100 ras-related protein Rab-2 (RAB2) 7 39.9 6 rRNA-processing protein EBP2, putative PF3D7_1028300 (EBP2) 13 39.7 17 conserved Plasmodium protein, unknown PF3D7_1006700 function 9 39.7 7 PF3D7_0903900 60S ribosomal protein L32 (RPL32) 6 39.7 7 PF3D7_1116200.1;PF pyridoxine biosynthesis protein PDX2 3D7_1116200.2 (PDX2) 6 39.7 5 high molecular weight rhoptry protein 3 PF3D7_0905400 (RhopH3) 26 39.6 57 PF3D7_1004000 60S ribosomal protein L13, putative 10 39.6 10 PF3D7_0415900 60S ribosomal protein L15, putative 11 39.5 14 PF3D7_1138500 protein phosphatase 2C (PP2C) 29 39.4 44 PF3D7_1104400 conserved protein, unknown function 18 39.2 17 bifunctional dihydrofolate reductase- PF3D7_0417200 thymidylate synthase (DHFR-TS) 20 39 13 PF3D7_1213800 proline--tRNA ligase (PRS) 22 38.9 26 PF3D7_0717700 serine--tRNA ligase, putative 13 38.8 20 eukaryotic translation initiation factor 2 PF3D7_0728000 alpha subunit, putative 11 38.6 6 PF3D7_1142500 60S ribosomal protein L28 (RPL28) 5 38.6 6 PF3D7_1252100 rhoptry neck protein 3 (RON3) 66 38.4 89 PF3D7_0930300;PF3D 7_1312000 merozoite surface protein 1 (MSP1) 57 38.3 54 26S protease regulatory subunit 8, putative PF3D7_1248900 (RPT6) 12 38.2 5 PF3D7_0306900 40S ribosomal protein S23, putative 5 37.9 5 191

PF3D7_1020700 histone acetyltransferase, putative 34 37.7 36 PF3D7_0612900 nucleolar GTP-binding protein 1, putative 19 37.6 21 cytoadherence linked asexual protein 9 PF3D7_0935800 (CLAG9) 43 37.5 66 PF3D7_1351400 60S ribosomal protein L17, putative 8 37.4 15 PF3D7_1027300 peroxiredoxin (nPrx) 25 37.2 58 PF3D7_1352500 thioredoxin-related protein, putative 8 37 9 conserved Plasmodium protein, unknown PF3D7_1419700 function 13 36.9 22 conserved Plasmodium protein, unknown PF3D7_1205600 function 10 36.9 39 PF3D7_0201900 erythrocyte membrane protein 3 (EMP3) 8 36.8 0 PF3D7_1109900 60S ribosomal protein L36 (RPL36) 5 36.6 5 PF3D7_0210100.1 60S ribosomal protein L37ae, putative 4 36.5 4 PF3D7_0706000 importin-7, putative 33 36.3 43 eukaryotic translation initiation factor 3, PF3D7_0528200 subunit 6, putative 13 36 13 rab specific GDP dissociation inhibitor PF3D7_1242800 (rabGDI) 10 35.9 12 26S proteasome regulatory subunit RPN11, PF3D7_1368100 putative (RPN11) 6 35.7 4 PF3D7_0921000.1;PF 3D7_0921000.2 ubiquitin-conjugating enzyme, putative 3 35.6 2 PF3D7_0801800 mannose-6-phosphate isomerase, putative 27 35.5 32 PF3D7_0714000 histone H2B variant (H2B.Z) 6 35 9 PF3D7_1108700 heat shock protein DnaJ homologue Pfj2 15 34.8 22 nascent polypeptide associated complex PF3D7_0621800 alpha chain, putative 3 34.8 4 golgi organization and biogenesis factor, PF3D7_0418200 putative 11 34.7 12 PF3D7_0519400 40S ribosomal protein S24 (RPS24) 7 34.6 4 ribonucleoside-diphosphate reductase, PF3D7_1437200 large subunit, putative 22 34.4 20 PF3D7_0810800 dihydropteroate synthetase (DHPS) 17 34.1 18 PF3D7_0211800 asparagine--tRNA ligase (AsnRS) 11 34.1 17 cAMP-dependent protein kinase regulatory PF3D7_1223100 subunit (PKAr) 13 34 26 PF3D7_1217900 conserved protein, unknown function 9 34 14 conserved Plasmodium protein, unknown PF3D7_1105800 function 5 33.8 11 PF3D7_1342600 myosin A (MyoA) 15 33.7 28 conserved Plasmodium protein, unknown PF3D7_0813300 function 11 33.7 18 PF3D7_1441200 60S ribosomal protein L1, putative 10 33.6 6 conserved Plasmodium protein, unknown PF3D7_1459400 function 10 33.5 15 ribonucleotide reductase small subunit, PF3D7_1015800 putative 10 33.4 20 192

PF3D7_1360900 polyadenylate-binding protein, putative 7 33.4 11 PF3D7_1129400 RNA methyltransferase, putative 28 33.3 39 PF3D7_0312800 60S ribosomal protein L26, putative 4 33.3 6 eukaryotic translation initiation factor 3 PF3D7_1206200 subunit 8, putative 26 33.2 40 conserved Plasmodium protein, unknown PF3D7_1128900 function 13 33 6 conserved Plasmodium protein, unknown PF3D7_0606000 function 26 32.9 9 PF3D7_1134100;PF3D 7_0710000 protein disulfide isomerase (PDI-11) 12 32.9 11 PF3D7_0321500 peptidase, putative 30 32.8 32 conserved Plasmodium protein, unknown PF3D7_0710700 function 10 32.8 18 PF3D7_0719700 40S ribosomal protein S10, putative 5 32.8 6 PF3D7_0304400 60S ribosomal protein L44 (RPL44) 8 32.7 13 eukaryotic translation initiation factor 3 PF3D7_0716800 37.28 kDa subunit, putative 7 32.7 9 large ribosomal subunit processing factor, PF3D7_1126400 putative 3 32.6 0 PF3D7_1405900 RNA-binding protein, putative 31 32.5 28 mature parasite-infected erythrocyte surface antigen,erythrocyte membrane PF3D7_0500800 protein 2 (MESA) 36 32.4 25 signal recognition particle subunit SRP68, PF3D7_0621900 putative (SRP68) 18 32.3 21 PF3D7_0929200 RNA-binding protein, putative 7 32.2 9 conserved Plasmodium protein, unknown PF3D7_1361800 function 55 32.1 84 conserved Plasmodium protein, unknown PF3D7_0308100 function 43 32.1 43 conserved Plasmodium protein, unknown PF3D7_0828300 function 19 32.1 41 PF3D7_1471100 exported protein 2 (EXP2) 6 32.1 6 PF3D7_1445200 RNA helicase, putative 19 31.8 18 PF3D7_1021500 RNA helicase, putative 15 31.6 16 PF3D7_0815200 importin beta, putative 20 31.5 22 conserved Plasmodium protein, unknown PF3D7_1238700 function 13 31.5 10 PF3D7_1309100 60S ribosomal protein L24, putative 7 31.5 8 PF3D7_0705700 40S ribosomal protein S29, putative 3 31.5 3 PF3D7_1420400 glycine--tRNA ligase (GlyRS) 23 31.4 22 PF3D7_0710600 60S ribosomal protein L34 (RPL34) 7 31.3 30 PF3D7_1460300 60S ribosomal protein L29, putative 3 31.3 11 PF3D7_0817700 rhoptry neck protein 5 (RON5) 27 31.2 46 choline/ethanolaminephosphotransferase, PF3D7_0628300 putative (CEPT) 8 31.2 9 subunit of proteaseome activator complex, PF3D7_0907700 putative 6 31.2 5 193

PF3D7_0807900 tyrosine--tRNA ligase (TyrRS) 8 31.1 7 nuclear movement protein, putative PF3D7_1336800 (NUDC) 12 30.8 20 knob-associated histidine-rich protein PF3D7_0202000 (KAHRP) 24 30.6 2 PF3D7_1218600 arginine--tRNA ligase, putative 13 30.5 13 succinate dehydrogenase subunit 4, PF3D7_1010300 putative (SDH4) 5 30.5 4 PF3D7_1308200 carbamoyl phosphate synthetase (cpsSII) 49 30.3 76 autophagy-related protein 18, putative PF3D7_1012900 (ATG18) 10 30.3 10 mediator of RNA polymerase II PF3D7_0822100 transcription subunit 7, putative (MED7) 6 30.3 6 PF3D7_1238800 acyl-CoA synthetase (ACS11) 17 30.2 13 26S proteasome regulatory subunit p55, PF3D7_1017900 putative (RPN5) 9 30.2 8 conserved Plasmodium protein, unknown PF3D7_1359600 function 37 30 66 signal recognition particle receptor, beta PF3D7_1246800 subunit (SRPR-beta) 7 29.9 8 DNA replication licensing factor MCM3, PF3D7_0527000 putative (MCM3) 19 29.8 26 conserved Plasmodium protein, unknown PF3D7_1323900 function 8 29.8 13 PF3D7_1365900;PF3D 7_1211800 ubiquitin-60S ribosomal protein L40 3 29.7 9 PF3D7_0111500 UMP-CMP kinase, putative 8 29.6 10 rRNA processing and telomere maintaining PF3D7_0925200 methyltransferase, putative 10 29.5 4 PF3D7_1134000 heat shock protein 70 (HSP70-3) 18 29 19 PF3D7_1412500 actin II (ACT2) 6 29 7 PF3D7_1434300 Hsp70/Hsp90 organizing protein (HOP) 10 28.9 14 PF3D7_0102900 aspartate--tRNA ligase 16 28.8 17 PF3D7_1139900 conserved protein, unknown function 4 28.8 8 conserved Plasmodium protein, unknown PF3D7_1460500 function 32 28.6 47 PF3D7_1241700 replication factor C subunit 4, putative 7 28.6 6 PF3D7_0422700 eukaryotic initiation factor, putative 8 28.5 7 PF3D7_1323200 vacuolar ATP synthase subunit g, putative 4 28.5 5 PF3D7_1145400 dynamin-like protein (DYN1) 18 28.3 22 PF3D7_0419600 ran binding protein 1, putative 10 28.2 16 Plasmodium exported protein (PHISTb), PF3D7_0401800 unknown function (PfD80) 22 28 8 calponin homology domain-containing PF3D7_1447800 protein, putative 46 27.8 11 PF3D7_0303200 HAD superfamily protein, putative 26 27.8 107 eukaryotic translation initiation factor 3 PF3D7_1212700 subunit 10, putative 33 27.7 40 PF3D7_1306200 conserved Plasmodium protein, unknown 9 27.6 8 194

function PF3D7_0903700 alpha tubulin 1 8 27.6 22 PF3D7_0823200 RNA-binding protein, putative 5 27.6 5 PF3D7_0918000 secreted acid phosphatase (GAP50) 9 27.5 10 26S protease regulatory subunit 4, putative PF3D7_1008400 (RPT2) 8 27.5 10 PF3D7_0913200 elongation factor 1-beta (EF-1beta) 4 27.5 4 polyadenylate-binding protein, putative PF3D7_1224300 (PABP) 20 27.4 30 eukaryotic translation initiation factor 5A PF3D7_1204300 (EIF5A) 3 27.3 5 conserved Plasmodium protein, unknown PF3D7_1422400 function 37 27.2 59 ubiquitin-40S ribosomal protein S27a, PF3D7_1402500 putative 7 26.8 14 haloacid dehalogenase-like hydrolase, PF3D7_1226300 putative (HAD2) 6 26.8 6 glideosome associated protein with PF3D7_1406800 multiple membrane spans 3 (GAPM3) 6 26.8 6 PF3D7_1235600 serine hydroxymethyltransferase (SHMT) 12 26.7 5 PF3D7_0627700 transportin 24 26.6 40 PF3D7_1220900 heterochromatin protein 1 (HP1) 5 26.3 1 3-oxo-5-alpha-steroid 4-dehydrogenase, PF3D7_1135900 putative 7 26 6 PF3D7_1118200 heat shock protein 90, putative 15 25.8 13 PF3D7_1331700 glutamine--tRNA ligase, putative 14 25.8 10 1-acyl-sn-glycerol-3-phosphate PF3D7_1444300 acyltransferase, putative (LPAAT) 5 25.7 8 large subunit rRNA methyltransferase, PF3D7_1354300 putative 27 25.6 27 PF3D7_0518600 WD-repeat protein, putative 26 25.6 37 voltage-dependent anion-selective channel PF3D7_1432100 protein, putative 6 25.6 3 conserved Plasmodium protein, unknown PF3D7_0932800 function 26 25.5 29 PF3D7_0607000 translation initiation factor IF-2, putative 17 25.5 24 PF3D7_0527500 Hsc70-interacting protein (HIP) 10 25.5 16 26S proteasome regulatory subunit RPN6 PF3D7_1402300 (RPN6) 10 25.4 8 glutamate dehydrogenase, putative PF3D7_0802000 (GDH3) 24 25.3 22 conserved Plasmodium protein (10b PF3D7_1021900 antigen), unknown function 47 25.2 100 PF3D7_1225800 ubiquitin-activating enzyme E1 (UBA1) 16 25.2 43 26S proteasome regulatory subunit RPN3, PF3D7_1338100 putative (RPN3) 10 25.2 8 PF3D7_0302900 exportin-1, putative 26 25 29 PF3D7_0614500 60S ribosomal protein L19 (RPL19) 5 24.7 9 PF3D7_0310600.2;PF SAC3/GNAP family-related protein, 3 24.4 3 195

3D7_0310600.1 putative PF3D7_1318800 translocation protein SEC63 (SEC63) 13 24.3 12 PF3D7_1325100 phosphoribosylpyrophosphate synthetase 8 24.3 3 PF3D7_1311400 AP-1 complex subunit mu, putative 6 24.3 4 PF3D7_0903200 ras-related protein RAB7 (RAB7) 4 24.3 9 FACT complex subunit SPT16, putative PF3D7_0517400 (FACT-L) 22 24.1 19 PF3D7_0213100 heat shock protein 40, putative 4 24.1 6 PF3D7_0819100 BRIX domain, putative 4 24.1 1 conserved Plasmodium protein, unknown PF3D7_1227700 function 17 24 9 pyridoxine biosynthesis protein PDX1 PF3D7_0621200 (PDX1) 5 23.9 6 PF3D7_0206700 adenylosuccinate lyase (ASL) 8 23.8 7 ADP-ribosylation factor GTPase-activating PF3D7_1244600 protein, putative (ARFGAP) 4 23.8 4 beta subunit of coatomer complex, PF3D7_0905900 putative (SEC27) 15 23.6 15 PF3D7_0512700 orotate phosphoribosyltransferase (OPRT) 6 23.5 5 glutamine-dependent NAD(+) synthetase, PF3D7_0926700 putative (NADSYN) 15 23.4 12 PF3D7_0919000 nucleosome assembly protein (NAPS) 3 23.4 8 PF3D7_0106800 ras-related protein Rab-5C (RAB5c) 3 23.4 5 DNA mismatch repair protein MSH2, PF3D7_1427500 putative (MSH2-1) 15 23.3 16 PF3D7_1368200 RNAse L inhibitor protein, putative 9 23.3 18 PF3D7_1311800 M1-family alanyl aminopeptidase (M1AAP) 15 23.2 21 conserved Plasmodium protein, unknown PF3D7_0520200 function 10 23.2 75 glycerol-3-phosphate dehydrogenase, PF3D7_1216200 putative 8 23.2 7 serine/threonine protein phosphatase PP1 PF3D7_1414400 (PP1) 5 23 7 lysine-specific histone demethylase 1, PF3D7_1211600 putative (LSD1) 55 22.9 79 FACT complex subunit SSRP1, putative PF3D7_1441400 (FACT-S) 10 22.9 15 PF3D7_1106000 RuvB-like helicase 2 (RUVB2) 7 22.9 7 PF3D7_1145100 coatomer subunit gamma, putative 15 22.8 15 PF3D7_1350100 lysine--tRNA ligase (KRS1) 10 22.8 13 conserved Plasmodium protein, unknown PF3D7_0821000 function 9 22.8 13 PF3D7_1362200 RuvB-like helicase 3 (RUVB3) 8 22.8 21 PF3D7_1458400 aminodeoxychorismate lyase (ADCL) 5 22.8 8 PF3D7_0923900 RNA-binding protein, putative 4 22.8 4 PF3D7_1219100 clathrin heavy chain, putative 33 22.7 43 conserved Plasmodium protein, unknown PF3D7_0423300 function 12 22.7 15 196

peptide chain release factor subunit 1, PF3D7_0212300 putative 7 22.7 8 conserved Plasmodium protein, unknown PF3D7_1213200 function 4 22.7 56 PF3D7_1413900 DnaJ protein, putative 7 22.6 1 ribonucleotide reductase small subunit PF3D7_1405600 (RNR) 6 22.6 14 PF3D7_0934500 vacuolar ATP synthase subunit e, putative 3 22.6 5 PF3D7_0903400 DEAD/DEAH box helicase, putative 40 22.5 23 PF3D7_1445100 histidine--tRNA ligase, putative 19 22.5 14 translation elongation factor EF-1, subunit PF3D7_1123400 alpha, putative 8 22.5 10 small subunit rRNA processing factor, PF3D7_1225500 putative 8 22.3 8 PF3D7_1336900 tryptophan--tRNA ligase (WRS) 9 22.2 12 PF3D7_1203700 nucleosome assembly protein (NAPL) 4 22.2 12 PF3D7_1463200 replication factor C3, putative (RFC3) 5 22.1 5 conserved Plasmodium protein, unknown PF3D7_1411500 function 15 22 24 heat shock protein 101,chaperone protein PF3D7_1116800 ClpB2 (HSP101) 14 22 8 ATP-dependent RNA helicase DBP10, PF3D7_0827000 putative (DBP10) 19 21.9 20 conserved Plasmodium protein, unknown PF3D7_1468100 function 41 21.8 52 eukaryotic translation initiation factor PF3D7_1019000 subunit eIF2A, putative 40 21.8 60 PF3D7_0312400 glycogen synthase kinase 3 (GSK3) 6 21.8 9 PF3D7_1331300 signal peptidase 21 kDa subunit (SP21) 5 21.7 3 eukaryotic translation initiation factor, PF3D7_0815600 putative 3 21.6 6 cell division cycle protein 48 homologue, PF3D7_0619400 putative 12 21.5 15 PF3D7_0113000 glutamic acid-rich protein (GARP) 9 21.4 32 26S proteasome regulatory subunit RPN12, PF3D7_0312300 putative (RPN12) 3 21.4 4 conserved Plasmodium protein, unknown PF3D7_1141300 function 18 21.3 16 translation initiation factor EIF-2b alpha PF3D7_0828500 subunit, putative 5 21.3 3 multiprotein bridging factor type 1, PF3D7_1128200 putative (MBF1) 3 21.3 8 PF3D7_1426200 arginine methyltransferase 1 (PRMT1) 6 21.2 4 vesicle-associated membrane protein, PF3D7_1439800 putative 3 21.2 7 PF3D7_0517300 pre-mRNA-splicing factor (SR1) 6 21.1 7 PF3D7_1410200 cytidine triphosphate synthetase 14 21 18 PF3D7_0523000 multidrug resistance protein (MDR1) 23 20.9 25 PF3D7_1210600 conserved Plasmodium protein, unknown 22 20.8 25 197

function PF3D7_1417800;PF3D DNA replication licensing factor MCM2 7_0525600 (MCM2) 15 20.7 22 shewanella-like protein phosphatase 1, PF3D7_1469200 putative (SHLP1) 6 20.7 1 PF3D7_1142600 60S ribosomal protein L35ae, putative 4 20.7 4 PF3D7_1333400 conserved protein, unknown function 4 20.6 3 conserved Plasmodium protein, unknown PF3D7_1138000 function 54 20.5 26 conserved Plasmodium protein, unknown PF3D7_0111800 function 12 20.5 11 PF3D7_0106100 vacuolar ATP synthase subunit c, putative 8 20.4 7 conserved Plasmodium protein, unknown PF3D7_1138700 function 32 20.3 41 PF3D7_1419100 ATP-dependent RNA helicase, putative 16 20.3 11 PF3D7_0316300.1;PF 3D7_0316300.2 inorganic pyrophosphatase, putative 4 20.3 7 conserved Plasmodium protein, unknown PF3D7_1226400 function 11 20.2 24 PF3D7_0823300 histone acetyltransferase GCN5 (GCN5) 20 20.1 27 conserved Plasmodium protein, unknown PF3D7_0105200 function 10 20.1 10 protein transport protein SEC61 subunit PF3D7_1346100 alpha (SEC61) 7 20.1 8 PF3D7_0528800 nucleolar preribosomal GTPase, putative 7 20.1 6 RNA-binding protein musashi, putative PF3D7_0916700 (HoMu) 7 20.1 6 deoxyribodipyrimidine photo-lyase, PF3D7_0513600 putative 18 20 29 PF3D7_0622800 leucine--tRNA ligase, putative 17 20 19 PF3D7_0829200 prohibitin, putative 3 19.9 0 conserved Plasmodium protein, unknown PF3D7_0407700 function 21 19.8 23 PF3D7_0106300 calcium-transporting ATPase (ATP6) 16 19.8 29 cochaperone prefoldin complex subunit, PF3D7_1128100 putative 4 19.8 6 PF3D7_0218000 replication factor C subunit 2, putative 5 19.7 6 Plasmodium exported protein (PHISTc), PF3D7_0936800 unknown function 6 19.6 4 conserved Plasmodium protein, unknown PF3D7_1447700 function 5 19.6 5 Leu/Phe-tRNA protein transferase, PF3D7_0212900 putative 4 19.5 7 isocitrate dehydrogenase (NADP), PF3D7_1345700 mitochondrial precursor (IDH) 7 19.4 7 conserved Plasmodium protein, unknown PF3D7_1332200 function 16 19.3 11 serine/arginine-rich splicing factor 4 PF3D7_1022400 (SRSF4) 8 19.3 12 PF3D7_0831400 Plasmodium exported protein, unknown 8 19.2 5 198

function PF3D7_1401800 choline kinase (CK) 7 18.9 13 conserved Plasmodium protein, unknown PF3D7_0108300 function 34 18.7 59 PF3D7_1205900 conserved protein, unknown function 15 18.7 21 small subunit rRNA processing factor, PF3D7_1451900 putative 12 18.7 18 PF3D7_1306600 vacuolar ATP synthase subunit h, putative 7 18.7 9 conserved Plasmodium protein, unknown PF3D7_0907300 function 4 18.6 6 PF3D7_1029600 adenosine deaminase (ADA) 4 18.5 6 PF3D7_0910100 Ran-binding protein, putative 20 18.4 18 U3/U14 snoRNA-associated small subunit PF3D7_1109400 rRNA processing protein, putative 5 18.1 7 PF3D7_1436000 glucose-6-phosphate isomerase (GPI) 9 18 7 glideosome associated protein with PF3D7_0423500 multiple membrane spans 2 (GAPM2) 3 18 2 PF3D7_1228600 merozoite surface protein 9 (MSP9) 12 17.9 10 PF3D7_1442300 tRNA binding protein, putative 6 17.9 5 PF3D7_0803700 tubulin gamma chain (g-tub) 6 17.9 5 FK506-binding protein (FKBP)-type PF3D7_1247400 peptidyl-prolyl isomerase (FKBP35) 4 17.8 4 conserved Plasmodium protein, unknown PF3D7_1456700 function 9 17.7 15 PF3D7_0624600 SNF2 helicase, putative (ISWI) 36 17.6 67 DNA replication licensing factor MCM4 PF3D7_1317100 (MCM4) 13 17.5 16 DNA replication licensing factor MCM7, PF3D7_0705400 putative (MCM7) 11 17.5 6 transmembrane protein Tmp21 PF3D7_1333300 homologue, putative 3 17.5 1 conserved Plasmodium protein, unknown PF3D7_0710200 function 39 17.4 117 ubiquitin carboxyl-terminal hydrolase, PF3D7_0527200 putative 8 17.2 9 PF3D7_0914700 transporter, putative 6 17.1 4 PF3D7_1342400 casein kinase II beta chain (CK2beta2) 4 17.1 4 PF3D7_0526200.2;PF ADP-ribosylation factor GTPase-activating 3D7_0526200.1 protein, putative (ARF-GAP) 7 17 11 phosphoenolpyruvate carboxykinase PF3D7_1342800 (PEPCK) 6 17 9 conserved Plasmodium protein, unknown PF3D7_0629600 function 4 17 5 conserved Plasmodium protein, unknown PF3D7_0819600 function 3 17 4 PF3D7_1340700 ras-related protein Rab-11B (RAB11b) 3 17 1 conserved Plasmodium protein, unknown PF3D7_1031300 function 11 16.9 4 PF3D7_1404500 rRNA biogenesis protein RRP5, putative 6 16.9 7 199

(RRP5) conserved Plasmodium protein, unknown PF3D7_0914100 function 19 16.8 17 26S proteasome regulatory subunit RPN10, PF3D7_0807800 putative (RPN10) 5 16.8 6 SNAP protein (soluble N-ethylmaleimide- sensitive factor attachment protein), PF3D7_0509000 putative 3 16.8 1 PF3D7_0606700 coatomer alpha subunit, putative 18 16.6 25 parasite-infected erythrocyte surface PF3D7_0310400 protein (PIESP1) 14 16.6 15 PF3D7_0605100 RNA-binding protein, putative 8 16.6 10 PF3D7_0629200 DnaJ protein, putative 6 16.6 4 PF3D7_0933200 calcyclin binding protein, putative 3 16.5 7 conserved Plasmodium protein, unknown PF3D7_0630600 function 12 16.4 14 conserved Plasmodium protein, unknown PF3D7_1249100 function 5 16.3 6 PF3D7_0413500 phosphoglucomutase-2 (PGM2) 3 16.3 3 calcium-dependent protein kinase 1 PF3D7_0217500 (CDPK1) 7 16.2 9 conserved Plasmodium protein, unknown PF3D7_1244700 function 3 16.2 5 conserved Plasmodium protein, unknown PF3D7_0217900 function 7 16 11 PF3D7_1452000 rhoptry neck protein 2 (RON2) 25 15.9 60 mRNA-decapping enzyme 2, putative PF3D7_1308900 (DCP2) 16 15.9 15 26S protease regulatory subunit 6a, PF3D7_1130400 putative (RPT5) 4 15.9 15 PF3D7_1461900 valine--tRNA ligase, putative 11 15.7 16 Plasmodium exported protein (PHISTa), PF3D7_0402000 unknown function 7 15.7 6 PF3D7_1026900 biotin--acetyl-CoA-carboxylase, putative 4 15.6 2 PF3D7_1014300 conserved protein, unknown function 31 15.5 53 PF3D7_1438400 metacaspase-like protein (MCA2) 24 15.5 14 PF3D7_1436600 cGMP-dependent protein kinase (PKG) 8 15.5 8 PF3D7_1218300 AP-3 complex subunit mu, putative 6 15.5 4 Plasmodium exported protein (PHISTb), PF3D7_0424600 unknown function 3 15.5 2 PF3D7_1226600 proliferating cell nuclear antigen 2 (PCNA2) 3 15.5 2 snoRNA-associated small subunit rRNA PF3D7_1217200 processing protein, putative 12 15.4 8 PF3D7_0627800 acetyl-CoA synthetase, putative (ACS) 8 15.4 13 conserved Plasmodium protein, unknown PF3D7_1465200 function 4 15.4 6 PF3D7_1015600 heat shock protein 60 (HSP60) 6 15.2 0 conserved Plasmodium protein, unknown PF3D7_0912400 function 5 15.2 3 200

conserved Plasmodium protein, unknown PF3D7_1338700 function 5 15.1 2 PF3D7_1022000 RNA-binding protein, putative 15 15 7 PF3D7_1455500 AP-1 complex subunit gamma, putative 9 15 10 PF3D7_1473200 DnaJ protein, putative 5 15 6 conserved Plasmodium membrane protein, PF3D7_1409400 unknown function 4 14.9 11 PF3D7_0919100 DnaJ protein, putative 4 14.9 3 PF3D7_0925700 histone deacetylase (HDAC1) 3 14.7 3 conserved Plasmodium protein, unknown PF3D7_0510100 function 24 14.6 62 replication factor C subunit 5, putative PF3D7_1111100 (RFC5) 4 14.6 5 PF3D7_1426700 phosphoenolpyruvate carboxylase (PEPC) 12 14.5 20 conserved Plasmodium protein, unknown PF3D7_0724700 function 39 14.4 42 phosphopantetheine adenylyltransferase, PF3D7_0704700 putative (PPAT) 14 14.4 8 mRNA binding Pumilio-homology domain PF3D7_0621300 protein, putative 12 14.4 9 PF3D7_0704400 phosphoinositide-binding protein, putative 9 14.4 12 PF3D7_1307900 tripartite motif protein, putative 10 14.3 8 PF3D7_0320800 ATP-dependent RNA helicase DDX6 (DOZI) 5 14.3 7 rRNA processing WD-repeat protein, PF3D7_1237600 putative 5 14.3 7 PF3D7_1429800 coatamer beta subunit, putative 16 14.2 21 PF3D7_1459000 ATP-dependent RNA helicase DBP5 (DBP5) 7 14.2 9 glucose-6-phosphate dehydrogenase-6- PF3D7_1453800 phosphogluconolactonase (G6PDH) 9 14.1 11 PF3D7_1329100 myosin C (MyoC) 22 14 26 DNA replication licensing factor MCM6 PF3D7_1355100 (MCM6) 9 14 12 PF3D7_1343700 kelch protein K13 (K13) 8 13.9 5 PF3D7_0810600 RNA helicase, putative 11 13.8 17 26S proteasome regulatory subunit RPN2, PF3D7_1466300 putative (RPN2) 8 13.8 15 U5 small nuclear ribonuclear protein, PF3D7_1003800 putative 8 13.8 10 PF3D7_1439900 triosephosphate isomerase (TIM) 3 13.7 2 PF3D7_1118300 insulinase, putative 14 13.6 26 PF3D7_1134800 coatomer delta subunit, putative 6 13.6 6 PF3D7_0409800 zinc finger protein, putative 5 13.6 10 conserved Plasmodium protein, unknown PF3D7_1117900 function 16 13.5 20 AP endonuclease (DNA-[apurinic or PF3D7_0305600 apyrimidinic site] lyase), putative 7 13.5 8 PF3D7_1033700 bromodomain protein, putative 5 13.5 6 PF3D7_0523100 mitochondrial processing peptidase alpha 5 13.5 2 201

subunit, putative PF3D7_1438700 DNA primase small subunit 4 13.5 9 conserved Plasmodium protein, unknown PF3D7_0617200 function 4 13.5 2 eukaryotic translation initiation factor 3 PF3D7_1007900 subunit 7, putative 6 13.4 6 conserved Plasmodium protein, unknown PF3D7_0308700 function 5 13.4 1 PF3D7_1133800 RNA (uracil-5-)methyltransferase, putative 10 13.3 10 choline-phosphate cytidylyltransferase PF3D7_1316600 (CCT) 6 13.2 12 PF3D7_0321400 protein kinase, putative 5 13.2 5 PF3D7_1120500 tRNA nucleotidyltransferase, putative 7 13.1 8 PF3D7_0616800 malate:quinone oxidoreductase, putative 5 13.1 2 PF3D7_1129000 spermidine synthase (SpdSyn) 3 13.1 2 conserved Plasmodium protein, unknown PF3D7_1356100 function 9 12.9 18 PF3D7_0703500 erythrocyte membrane-associated antigen 19 12.7 31 PF3D7_0416400 histone acetyltransferase, putative (HAT1) 11 12.7 15 PF3D7_0204700 hexose transporter (HT) 4 12.7 3 PF3D7_0922100 ubiquitin-like protein, putative 15 12.6 20 PF3D7_0503600 myosin B (MyoB) 7 12.6 126 mitochondrial-processing peptidase PF3D7_0933600 subunit beta, putative (MAS1) 4 12.6 7 PF3D7_0306200 activator of Hsp90 ATPase (AHA1) 4 12.6 4 PF3D7_0219600 replication factor C subunit 1, putative 10 12.5 14 serine/threonine protein phosphatase 2A PF3D7_1430100 activator (PTPA) 3 12.5 3 DNA replication licensing factor MCM5, PF3D7_1211700 putative (MCM5) 8 12.4 8 PF3D7_1241900 tetratricopeptide repeat protein, putative 7 12.4 11 PF3D7_1232100 60 kDa chaperonin (CPN60) 5 12.4 1 CCR4-NOT transcription complex subunit 1, PF3D7_1103800 putative (NOT1) 27 12.3 48 PF3D7_0617100 AP-2 complex subunit alpha, putative 14 12.2 14 PF3D7_0904000 GTPase-activating protein, putative 3 12.2 3 PF3D7_1438100 secretory complex protein 62 (SEC62) 3 12.2 2 origin recognition complex subunit 1 PF3D7_1203000 (ORC1) 9 12.1 8 26S proteasome regulatory subunit RPN1, PF3D7_0205900 putative (RPN1) 7 12.1 8 serine/threonine protein kinase, FIKK PF3D7_0424500 family (FIKK4.1) 7 12.1 7 conserved Plasmodium protein, unknown PF3D7_0922800 function 39 12 37 pre-mRNA-splicing factor SYF1, putative PF3D7_1235900 (XAB2) 10 12 16 PF3D7_0825500 protein KRI1, putative (KRI1) 5 11.9 11 202

PF3D7_1412700 AAA family ATPase, putative 11 11.8 13 conserved Plasmodium protein, unknown PF3D7_1026000 function 3 11.8 10 N2,N2-dimethylguanosine tRNA PF3D7_1319300 methyltransferase, putative 6 11.7 7 PF3D7_1230700 protein transport protein SEC13 (SEC13) 6 11.7 5 conserved Plasmodium protein, unknown PF3D7_1442400 function 20 11.5 22 gamma-glutamylcysteine synthetase PF3D7_0918900 (gammaGCS) 9 11.5 15 phospholipid-transporting ATPase, PF3D7_1223400 putative 14 11.4 15 PF3D7_0920800 inosine-5-monophosphate dehydrogenase 4 11.4 4 conserved Plasmodium protein, unknown PF3D7_1445700 function 3 11.4 4 conserved Plasmodium protein, unknown PF3D7_0321100 function 15 11.2 18 S-adenosylmethionine decarboxylase/ornithine decarboxylase PF3D7_1033100 (AdoMetDC/ODC) 14 11.2 23 PF3D7_0629800 cullin-like protein, putative 7 11.2 7 DNA mismatch repair protein MSH6, PF3D7_0505500 putative (MSH6) 11 11 13 conserved Plasmodium protein, unknown PF3D7_0817600 function 6 11 3 signal recognition particle receptor alpha PF3D7_1366600 subunit (SRPR-alpha) 5 10.9 5 conserved Plasmodium protein, unknown PF3D7_1208900 function 10 10.8 15 vacuolar proton translocating ATPase PF3D7_0806800 subunit A, putative 7 10.8 7 nicotinate phosphoribosyltransferase, PF3D7_0629100 putative (NAPRT) 5 10.8 10 chromatin assembly factor 1 subunit, PF3D7_1329300 putative 4 10.8 12 conserved Plasmodium protein, unknown PF3D7_1207000 function 23 10.7 14 cytosolic preribosomal GTP-binding PF3D7_0524400 protein, putative 4 10.7 2 DEAD/DEAH box ATP-dependent RNA PF3D7_0521700 helicase, putative 7 10.6 8 PF3D7_0515000 RNA-binding protein, putative 5 10.6 9 PF3D7_0321600 ATP-dependent RNA helicase, putative 4 10.6 29 PF3D7_0931000 elongation factor Tu, putative 5 10.5 5 nucleolar rRNA processing protein, PF3D7_0722600 putative 4 10.5 12 transcription factor with AP2 domain(s) PF3D7_0613800 (ApiAP2) 27 10.4 49 phenylalanyl-tRNA synthetase beta chain, PF3D7_1104000 putative 5 10.4 8 203

conserved Plasmodium protein, unknown PF3D7_0619700 function 5 10.4 5 26S proteasome regulatory subunit RPN9, PF3D7_1030500 putative (RPN9) 5 10.4 3 PF3D7_1004400 RNA-binding protein, putative 7 10.3 10 PF3D7_1005500 regulator of nonsense transcripts, putative 10 10.2 21 PF3D7_1347200 nucleoside transporter 1 (NT1) 3 10.2 2 pre-mRNA-processing-splicing factor 8, PF3D7_0405400 putative (PRPF8) 20 10.1 31 transcription factor with AP2 domain(s), PF3D7_1449500 putative (ApiAP2) 6 10.1 7 N-ethylmaleimide-sensitive fusion protein PF3D7_0303000 (NSF) 6 10.1 4 eukaryotic translation initiation factor PF3D7_1438000 eIF2A, putative 4 10.1 9 PF3D7_0720400 ferrodoxin reductase-like protein 4 10.1 5 Plasmodium exported protein (PHISTa), PF3D7_1372000 unknown function 3 10.1 0 PF3D7_1008000 histone deacetylase 2 (HDA2) 20 10 29 conserved Plasmodium protein, unknown PF3D7_1462300 function 8 10 10 conserved Plasmodium protein, unknown PF3D7_0813400 function 7 10 6 Plasmodium exported protein (PHISTb), PF3D7_1401600 unknown function 4 10 2 PF3D7_0214100 protein transport protein SEC31 (SEC31) 9 9.9 16 PF3D7_0304200 EH (Eps15 homology) protein (PAST1) 3 9.9 5 conserved Plasmodium protein, unknown PF3D7_0210900 function 3 9.9 3 conserved Plasmodium protein, unknown PF3D7_1247700 function 3 9.8 3 PF3D7_1419800.1;PF 3D7_1419800.2 glutathione reductase (GR) 3 9.8 1 PF3D7_0314700 zinc finger protein, putative 7 9.7 12 PF3D7_0629400 RNA-binding protein, putative 5 9.7 6 conserved Plasmodium protein, unknown PF3D7_0306100 function 3 9.7 22 conserved Plasmodium protein, unknown PF3D7_0811600 function 8 9.6 10 PF3D7_1147500 farnesyltransferase beta subunit, putative 7 9.6 16 Plasmodium exported protein (PHISTc), PF3D7_0801000 unknown function 7 9.6 0 PF3D7_0804900 GTPase-activating protein, putative 3 9.6 4 conserved Plasmodium protein, unknown PF3D7_1330800 function 3 9.6 2 conserved Plasmodium protein, unknown PF3D7_1142100 function 18 9.5 23 conserved Plasmodium protein, unknown PF3D7_1248700 function 15 9.5 25 PF3D7_0805700 serine/threonine protein kinase, FIKK 11 9.5 19 204

family (FIKK8) cytoadherence linked asexual protein 8 PF3D7_0831600 (CLAG8) 7 9.5 8 PF3D7_1315900 exportin-1, putative 6 9.5 11 PF3D7_1034000 Sec1 family protein, putative 4 9.5 1 large ribosomal subunit nuclear export PF3D7_1457700 factor, putative 11 9.3 2 PF3D7_1115400 cysteine proteinase falcipain 3 (FP3) 4 9.3 4 cell differentiation protein, putative PF3D7_0507600 (CAF40) 4 9.2 11 PF3D7_1435700.2;PF 3D7_1435700.1 ataxin-2 like protein, putative 5 9.1 11 DEAD/DEAH box ATP-dependent RNA PF3D7_1251500 helicase, putative 5 9.1 4 PF3D7_0217100 ATP synthase F1, alpha subunit 5 9.1 5 conserved Plasmodium protein, unknown PF3D7_0507800 function 10 9 15 PF3D7_1321700 splicing factor 1 (SF1) 5 9 4 PF3D7_0934700 UBX domain, putative 4 9 4 cytoadherence linked asexual protein 2 PF3D7_0220800 (CLAG2) 4 9 4 conserved Plasmodium protein, unknown PF3D7_0813000 function 3 9 6 polypyrimidine tract binding protein, PF3D7_0606500 putative 3 9 3 conserved Plasmodium protein, unknown PF3D7_1243400 function 3 9 0 conserved Plasmodium protein, unknown PF3D7_0707200 function 13 8.9 15 conserved Plasmodium membrane protein, PF3D7_1419400 unknown function 12 8.9 30 PF3D7_1466900 rhoptry protein, putative 7 8.9 11 inorganic anion exchanger, inorganic anion PF3D7_1471200 antiporter (SulP) 4 8.9 7 26S proteasome regulatory subunit RPN7, PF3D7_1129200 putative (RPN7) 4 8.9 3 splicing factor 3A subunit 1, putative PF3D7_1474500 (SF3A1) 5 8.8 15 conserved Plasmodium protein, unknown PF3D7_0111400 function (GEXP19) 5 8.8 6 histone S-adenosyl methyltransferase, PF3D7_1227800 putative 9 8.7 19 DNA-directed RNA polymerase II second PF3D7_0215700 largest subunit, putative 8 8.7 13 conserved Plasmodium membrane protein, PF3D7_0721000 unknown function 19 8.6 20 conserved Plasmodium protein, unknown PF3D7_0706500 function 10 8.6 17 nucleoporin NUP116/NSP116, putative PF3D7_1473700 (NUP116) 8 8.6 5 205

pre-mRNA-splicing factor ATP-dependent PF3D7_1231600 RNA helicase PRP2, putative (PRP2) 6 8.6 7 conserved Plasmodium membrane protein, PF3D7_1137300 unknown function 5 8.6 5 protein geranylgeranyltransferase type II, PF3D7_1242600 alpha subunit, putative 4 8.6 11 conserved Plasmodium protein, unknown PF3D7_0218200 function 4 8.6 6 PF3D7_1334200 chaperone binding protein, putative 3 8.6 8 ubiquitin carboxyl-terminal hydrolase, PF3D7_0726500 putative 16 8.5 33 PF3D7_1433500 DNA topoisomerase II, putative 9 8.5 15 splicing factor 3B subunit 3, putative PF3D7_1234800 (SF3B3) 7 8.5 15 root hair defective 3 GTP-binding protein PF3D7_1416100 (RHD3) homolog, putative 5 8.5 9 PF3D7_1347100 DNA topoisomerase 3, putative (TOP3) 5 8.5 8 PF3D7_0309500 asparagine synthetase, putative 4 8.5 1 PF3D7_1361100 protein transport protein Sec24A (SEC24A) 5 8.4 10 PF3D7_0811400 conserved protein, unknown function 3 8.4 6 conserved Plasmodium protein, unknown PF3D7_0407800 function 11 8.3 33 PF3D7_1139100 RNA-binding protein, putative 11 8.3 16 PF3D7_0731600 acyl-CoA synthetase (ACS5) 4 8.3 1 conserved Plasmodium protein, unknown PF3D7_1014900 function 16 8.2 70 transcription factor with AP2 domain(s) PF3D7_1007700 (ApiAP2) 12 8.2 22 conserved Plasmodium protein, unknown PF3D7_0813500 function 3 8.2 9 P-loop containing nucleoside triphosphate PF3D7_1352700 hydrolase, putative 13 8.1 23 PF3D7_1330600 elongation factor Tu, putative 3 8.1 3 multidrug resistance-associated protein 1 PF3D7_0112200 (MRP1) 10 8 13 conserved Plasmodium protein, unknown PF3D7_0408100 function 7 8 7 DEAD/DEAH box ATP-dependent RNA PF3D7_0218400 helicase, putative 3 8 4 PF3D7_0728600 zinc finger, C3HC4 type, putative 15 7.9 29 eukaryotic translation initation factor 4 PF3D7_1312900 gamma, putative (EIF4G) 8 7.9 15 secreted ookinete protein, putative PF3D7_0108700 (PSOP24) 5 7.9 14 ATP-dependent protease ATPase subunit PF3D7_0907400 ClpY (ClpY) 3 7.9 5 PF3D7_1131100 serpentine receptor, putative (SR1) 3 7.9 4 pre-mRNA-splicing factor ATP-dependent PF3D7_0917600 RNA helicase PRP43, putative (PRP43) 3 7.9 2 PF3D7_1454400 aminopeptidase P (APP) 3 7.9 1 206

conserved Plasmodium protein, unknown PF3D7_1369500 function 5 7.8 4 asparagine and aspartate rich protein 1 PF3D7_1233600 (AARP1) 27 7.7 15 DNA-directed RNA polymerase II subunit PF3D7_0318200 RPB1, putative (RPB1) 11 7.7 22 conserved Plasmodium protein, unknown PF3D7_1126500 function 7 7.7 10 PF3D7_1126000 threonine--tRNA ligase (ThrRS) 5 7.7 10 conserved Plasmodium protein, unknown PF3D7_1246300 function 4 7.7 10 conserved Plasmodium protein, unknown PF3D7_1230800 function 3 7.7 12 ubiquitin carboxyl-terminal hydrolase 1, PF3D7_0104300 putative (UBP1) 19 7.6 30 PF3D7_1353400 Ran-binding protein, putative 7 7.6 23 spindle assembly abnormal protein 4, PF3D7_1458500 putative (SAS4) 7 7.6 1 conserved Plasmodium protein, unknown PF3D7_1468900 function 5 7.6 3 Plasmodium exported protein (PHISTb), PF3D7_1476300 unknown function 4 7.5 2 conserved Plasmodium protein, unknown PF3D7_1455300 function 3 7.5 2 PF3D7_0704600 E3 ubiquitin-protein ligase (UT) 20 7.4 43 non-SERCA-type Ca2+ -transporting P- PF3D7_1211900 ATPase (ATP4) 5 7.4 11 PF3D7_0709000 chloroquine resistance transporter (CRT) 3 7.3 4 eukaryotic translation initiation factor 5, PF3D7_1206700 putative 3 7.3 4 peripheral plastid protein 1, putative PF3D7_1212100 (PPP1) 3 7.3 0 PF3D7_0824400 nucleoside transporter 2 (NT2) 3 7.2 6 PF3D7_1037500 dynamin-like protein (DYN2) 3 7.1 7 PF3D7_1135100 protein phosphatase 2C, putative 3 7.1 1 PF3D7_0629700 SET domain protein, putative (SET1) 37 7 34 NLI interacting factor-like phosphatase, PF3D7_1012700 putative (NIF4) 7 7 29 PF3D7_1121700 protein GCN20 (GCN20) 4 7 6 conserved Plasmodium protein, unknown PF3D7_1206800 function 4 7 3 glycerol-3-phosphate 1-O-acyltransferase PF3D7_1212500 (GAT) 3 7 0 conserved Plasmodium protein, unknown PF3D7_0529800 function 10 6.9 2 PF3D7_1134500 alpha/beta hydrolase, putative 8 6.9 9 PF3D7_0528100 AP-1 complex subunit beta, putative 5 6.9 11 conserved Plasmodium protein, unknown PF3D7_1013300 function 5 6.8 1 PF3D7_0705300 origin recognition complex subunit 2, 4 6.8 3 207

putative (ORC2) PF3D7_1251200 coronin 3 6.8 6 PF3D7_1437400 pantothenate kinase, putative 3 6.8 4 conserved Plasmodium protein, unknown PF3D7_0621100 function 6 6.7 9 PF3D7_0519800 conserved protein, unknown function 3 6.7 1 conserved Plasmodium protein, unknown PF3D7_1314100 function 4 6.6 10 conserved Plasmodium protein, unknown PF3D7_1366900 function 3 6.6 4 PF3D7_0207600 serine repeat antigen 5 (SERA5) 4 6.5 4 PF3D7_1142700 methyltransferase, putative 3 6.5 1 PF3D7_0110500 bromodomain protein, putative 10 6.4 36 multidrug resistance protein 2 (heavy PF3D7_1447900 metal transport family) (MDR2) 5 6.4 4 PF3D7_0302100 serine/threonine protein kinase (SRPK1) 5 6.4 1 PF3D7_1446200 M17 leucyl aminopeptidase (LAP) 3 6.4 6 PF3D7_1423700;REV_ conserved Plasmodium protein, unknown _PF3D7_1114900 function 9 6.3 25 conserved Plasmodium protein, unknown PF3D7_1213900 function 5 6.3 5 conserved Plasmodium protein, unknown PF3D7_1412100 function 4 6.3 18 ethanolamine-phosphate PF3D7_1347700 cytidylyltransferase (ECT) 3 6.3 3 PF3D7_0612200 leucine-rich repeat protein (LRR6) 8 6.2 3 PF3D7_1129900 transporter, putative 3 6.2 4 conserved Plasmodium protein, unknown PF3D7_1409600 function 3 6.2 3 PF3D7_0511500 RNA pseudouridylate synthase, putative 44 6.1 51 PF3D7_1212900 bromodomain protein, putative 5 6.1 6 PF3D7_1344200 heat shock protein 110, putative (HSP110) 5 6.1 1 glideosome-associated protein 40, putative PF3D7_0515700 (GAP40) 3 6.1 3 PF3D7_0907600 translation initiation factor SUI1, putative 3 6.1 3 ATP-dependent zinc metalloprotease FTSH PF3D7_1239700 1 (FTSH1) 3 6.1 2 chromodomain-helicase-DNA-binding PF3D7_1023900 protein 1 homolog, putative (CHD1) 16 6 37 PF3D7_1104200 chromatin remodeling protein (SNF2L) 6 6 16 conserved Plasmodium protein, unknown PF3D7_0824900 function 4 6 5 PF3D7_0613700 syntaxin binding protein, putative 3 6 3 PF3D7_1417200 NOT family protein, putative 18 5.9 35 PF3D7_0725000 exonuclease I, putative 5 5.9 23 conserved Plasmodium protein, unknown PF3D7_1345800 function 5 5.9 8 PF3D7_1017000 DNA polymerase delta catalytic subunit 4 5.9 5 208

PF3D7_1007200 rhoGAP GTPase, putative 3 5.9 2 PF3D7_1429900 ATP-dependent DNA helicase, putative 8 5.8 5 conserved Plasmodium protein, unknown PF3D7_0704800 function 7 5.8 10 ubiquitin conjugation factor E4 B, putative PF3D7_0826500 (UBE4B) 5 5.8 12 serine/threonine protein phosphatase 5 PF3D7_1355500 (PP5) 4 5.8 8 splicing factor 3A subunit 3, putative PF3D7_0924700 (SF3A3) 3 5.8 4 PF3D7_0926100 protein kinase, putative 18 5.7 40 conserved Plasmodium membrane protein, PF3D7_0903300 unknown function 10 5.6 24 PF3D7_0707700 E3 ubiquitin-protein ligase, putative 4 5.6 2 conserved Plasmodium protein, unknown PF3D7_0508900 function 11 5.5 12 conserved Plasmodium protein, unknown PF3D7_0729100 function 10 5.5 12 conserved Plasmodium protein, unknown PF3D7_1123000 function 9 5.4 8 PF3D7_0811300 CCR4-associated factor 1 (CAF1) 6 5.4 25 conserved Plasmodium protein, unknown PF3D7_1352400 function 6 5.4 8 conserved Plasmodium protein, unknown PF3D7_1305300 function 14 5.3 12 regulator of chromosome condensation, PF3D7_0919900 putative 9 5.3 16 histone-arginine methyltransferase, PF3D7_0811500 putative (CARM1) 3 5.3 12 transcription factor with AP2 domain(s) PF3D7_1466400 (ApiAP2) 3 5.3 9 conserved Plasmodium protein, unknown PF3D7_1312800 function 10 5.2 25 PF3D7_1239200;REV_ transcription factor with AP2 domain(s) _PF3D7_1348400 (ApiAP2) 9 5.2 65 pre-mRNA-splicing helicase BRR2, putative PF3D7_0422500 (BRR2) 9 5.2 17 PF3D7_1001600 alpha/beta hydrolase, putative 3 5.1 1 PF3D7_1344100 krox-like protein, putative 3 5 5

3D7attB Sample 1 Sequence MS/MS Protein IDs Product Description Peptides coverage Count [%] glyceraldehyde-3-phosphate dehydrogenase PF3D7_1462800 (GAPDH) 23 83.1 47 hypoxanthine-guanine PF3D7_1012400 phosphoribosyltransferase (HGPRT) 12 76.2 18 phosphoethanolamine N-methyltransferase PF3D7_1343000 (PMT) 14 70.3 41 209

PF3D7_1357100;PF3 D7_1357000 elongation factor 1-alpha 28 69.1 147 PF3D7_0922500 phosphoglycerate kinase (PGK) 28 68 59 PF3D7_1117700 GTP-binding nuclear protein RAN/TC4 (RAN) 16 66.4 37 PF3D7_1444800 fructose-bisphosphate aldolase (FBPA) 21 65.3 35 PF3D7_1224000 GTP cyclohydrolase I (GCH1) 24 62.7 45 PF3D7_0818900;PF3 D7_0831700 heat shock protein 70 (HSP70) 46 62.5 179 PF3D7_0422400 40S ribosomal protein S19 (RPS19) 11 61.8 15 PF3D7_0516200 40S ribosomal protein S11 7 61.6 9 PF3D7_1105400 40S ribosomal protein S4, putative 18 61.3 34 PF3D7_1015900 enolase (ENO) 20 60.8 28 PF3D7_0626800 pyruvate kinase (PyrK) 21 60.7 73 PF3D7_0608800 ornithine aminotransferase (OAT) 19 60.1 39 PF3D7_0317600 40S ribosomal protein S11, putative (RPS11) 12 59.6 18 PF3D7_0316800 40S ribosomal protein S15A, putative 8 59.2 13 PF3D7_1008700 tubulin beta chain 16 59.1 32 PF3D7_1105000 histone H4 (H4) 11 58.3 31 PF3D7_0617800 histone H2A (H2A) 5 57.6 16 PF3D7_1124600 ethanolamine kinase (EK) 20 57.4 38 PF3D7_0922200 S-adenosylmethionine synthetase (SAMS) 14 57.2 41 PF3D7_0510500 topoisomerase I (TopoI) 49 57.1 81 PF3D7_1323100 60S ribosomal protein L6, putative 7 56.8 11 PF3D7_1026800 40S ribosomal protein S2 (RPS2) 10 56.7 15 PF3D7_0610400 histone H3 (H3) 8 56.6 16 PF3D7_1424100 60S ribosomal protein L5, putative 17 55.4 28 PF3D7_0708400 heat shock protein 90 (HSP90) 39 55.2 586 PF3D7_1246200 actin I (ACT1) 15 54.8 43 PF3D7_1426000 60S ribosomal protein L21 (RPL21) 8 54.7 20 high molecular weight rhoptry protein 2 PF3D7_0929400 (RhopH2) 65 54.4 146 PF3D7_0322900 40S ribosomal protein S3A, putative 16 54.2 33 PF3D7_1460700 60S ribosomal protein L27 (RPL27) 9 54.1 14 PF3D7_1126200 40S ribosomal protein S18, putative 7 53.8 6 PF3D7_0617900 histone H3 variant, putative (H3.3) 3 53.7 7 PF3D7_0807300 ras-related protein Rab-18 (RAB18) 7 53.2 10 PF3D7_1338200 60S ribosomal protein L6-2, putative 10 52.9 19 PF3D7_0507100 60S ribosomal protein L4 (RPL4) 24 52.6 63 PF3D7_1414300 60S ribosomal protein L10, putative 12 52.1 36 PF3D7_0615400 ribonuclease, putative 154 52 224 box C/D snoRNP rRNA 2-O-methylation PF3D7_1118500 factor, putative 23 50.8 46 PF3D7_1447000 40S ribosomal protein S5 10 50.4 23 PF3D7_1341200 60S ribosomal protein L18, putative 12 49.5 20 PF3D7_0307200 60S ribosomal protein L7, putative 14 49.4 23 210

PF3D7_0320900 histone H2A variant, putative (H2A.Z) 6 49.4 6 PF3D7_1342000 40S ribosomal protein S6 26 49.3 76 PF3D7_1027800 60S ribosomal protein L3 (RPL3) 21 49.2 48 PF3D7_1019400 60S ribosomal protein L30e, putative 4 49.1 7 PF3D7_0714000 histone H2B variant (H2B.Z) 9 48.8 9 PF3D7_1130200 60S ribosomal protein P0 (PfP0) 10 48.4 15 PF3D7_1242700 40S ribosomal protein S17, putative 7 48.2 9 PF3D7_0524000 karyopherin beta (KASbeta) 38 48.1 91 PF3D7_1324900 L-lactate dehydrogenase (LDH) 11 47.5 14 PF3D7_0415900 60S ribosomal protein L15, putative 12 47.3 14 PF3D7_1451100 elongation factor 2 29 46.9 85 ATP-dependent RNA helicase UAP56 PF3D7_0209800 (UAP56) 15 46.8 40 PF3D7_1465900 40S ribosomal protein S3 15 46.2 28 PF3D7_1365900;PF3 D7_1211800 ubiquitin-60S ribosomal protein L40 5 45.3 9 PF3D7_1027300 peroxiredoxin (nPrx) 30 44.8 58 PF3D7_1317800 40S ribosomal protein S19 (RPS19) 5 44.8 17 PF3D7_1130100 60S ribosomal protein L38 (RPL38) 5 44.8 6 PF3D7_0618300 60S ribosomal protein L27a, putative 7 44.6 14 PF3D7_0813900 40S ribosomal protein S16, putative 9 44.4 14 PF3D7_1006800 RNA-binding protein, putative 9 43.9 17 PF3D7_1011800 PRE-binding protein (PREBP) 53 43.8 127 PF3D7_1468700 eukaryotic initiation factor 4A (eIF4A) 13 43.7 32 PF3D7_1408600 40S ribosomal protein S8e, putative 10 43.1 19 PF3D7_1410400 rhoptry-associated protein 1 (RAP1) 25 42.8 26 PF3D7_0321500 peptidase, putative 38 42.6 32 PF3D7_0903900 60S ribosomal protein L32 (RPL32) 5 42 7 PF3D7_1302800 40S ribosomal protein S7, putative 7 41.8 22 signal recognition particle subunit SRP72, PF3D7_1136400 putative (SRP72) 31 41.5 35 PF3D7_0823800 DnaJ protein, putative 17 40.9 34 PF3D7_0721600 40S ribosomal protein S5, putative 9 40.5 16 PF3D7_0624000 hexokinase (HK) 15 39.6 47 PF3D7_0826700 receptor for activated c kinase (RACK) 7 39.6 18 PF3D7_0814200 DNA/RNA-binding protein Alba 1 (ALBA1) 9 39.5 17 PF3D7_1136300 tudor staphylococcal nuclease (TSN) 37 39.3 64 PF3D7_0818200 14-3-3 protein (14-3-3I) 6 39.3 29 PF3D7_1424400 60S ribosomal protein L7-3, putative 13 39.2 28 high molecular weight rhoptry protein 3 PF3D7_0905400 (RhopH3) 27 39.1 57 PF3D7_0821700 60S ribosomal protein L22, putative 5 38.8 11 eukaryotic translation initiation factor 3 PF3D7_0612100 subunit L, putative 17 38.6 26 PF3D7_1105100 histone H2B (H2B) 7 38.5 34 PF3D7_1341300 60S ribosomal protein L18-2, putative 7 38.5 8 211

PF3D7_1358700 HVA22-like protein, putative 4 37.9 4 PF3D7_0517000 60S ribosomal protein L12, putative 5 37.6 4 PF3D7_1142500 60S ribosomal protein L28 (RPL28) 5 37 6 PF3D7_1347500 DNA/RNA-binding protein Alba 4 (ALBA4) 11 36.6 23 PF3D7_0516900 60S ribosomal protein L2 (RPL2) 10 36.5 18 PF3D7_0312800 60S ribosomal protein L26, putative 4 36.5 6 PF3D7_0501600 rhoptry-associated protein 2 (RAP2) 13 36.4 16 PF3D7_1302100 gamete antigen 27/25 (Pfg27) 5 36.4 13 PF3D7_0917900 heat shock protein 70 (HSP70-2) 17 36 42 PF3D7_1020900 ADP-ribosylation factor (ARF1) 5 35.4 15 PF3D7_1357800 TCP-1/cpn60 chaperonin family, putative 11 35.2 27 conserved Plasmodium protein, unknown PF3D7_1466800 function 26 35.1 46 cAMP-dependent protein kinase catalytic PF3D7_0934800 subunit (PKAc) 11 35.1 22 PF3D7_0416800 small GTP-binding protein sar1 (SAR1) 4 34.9 17 cytoadherence linked asexual protein 3.1 PF3D7_0302500 (CLAG3.1) 38 34.8 105 PF3D7_1120100 phosphoglycerate mutase, putative (PGM1) 7 34.8 18 PF3D7_1338300 elongation factor 1-gamma, putative 13 34.5 25 PF3D7_0606900 glutaredoxin-like protein (GLP2) 4 33.8 6 PF3D7_1136500.1;P F3D7_1136500.2 casein kinase 1 (CK1) 11 33.7 22 PF3D7_0406100 vacuolar ATP synthase subunit b 11 33.4 29 PF3D7_1003500 40S ribosomal protein S20e, putative 4 33.1 10 PF3D7_1025000 formin 2, putative 29 32.8 70 PF3D7_0719700 40S ribosomal protein S10, putative 4 32.8 6 PF3D7_1231100 ras-related protein Rab-2 (RAB2) 5 32.4 6 PF3D7_0520000 40S ribosomal protein S9, putative 6 32.3 8 PF3D7_0923900 RNA-binding protein, putative 4 32.2 4 PF3D7_0308200 TCP-1/cpn60 chaperonin family, putative 10 32.1 30 PF3D7_0814000 60S ribosomal protein L13-2, putative 8 32.1 18 PF3D7_1202900 high mobility group protein B1 (HMGB1) 3 32 7 PF3D7_1340300 nucleolar complex protein 2, putative 26 31.5 67 PF3D7_0706400 60S ribosomal protein L37 (RPL37) 4 31.5 6 PF3D7_1460300 60S ribosomal protein L29, putative 4 31.3 11 lysine-rich membrane-associated PHISTb PF3D7_0532400 protein (LyMP) 9 31.1 20 conserved Plasmodium protein, unknown PF3D7_0828300 function 17 31 41 PF3D7_1004000 60S ribosomal protein L13, putative 7 30.7 10 choline/ethanolaminephosphotransferase, PF3D7_0628300 putative (CEPT) 7 30.7 9 PF3D7_1311900 vacuolar ATP synthase subunit a (vapA) 9 30.6 24 PF3D7_1129400 RNA methyltransferase, putative 20 30.3 39 PF3D7_1349200 glutamate--tRNA ligase, putative 19 30.2 34 212

PF3D7_0519400 40S ribosomal protein S24 (RPS24) 4 30.1 4 PF3D7_1034900 methionine--tRNA ligase, putative 23 30 48 conserved Plasmodium protein, unknown PF3D7_0710700 function 6 29.9 18 PF3D7_1358800 40S ribosomal protein S15 (RPS15) 5 29.8 9 cytoadherence linked asexual protein 9 PF3D7_0935800 (CLAG9) 30 29.3 66 PF3D7_0501500 rhoptry-associated protein 3 (RAP3) 8 29.2 4 PF3D7_1323400 60S ribosomal protein L23 (RPL23) 8 28.9 22 rRNA-processing protein EBP2, putative PF3D7_1028300 (EBP2) 7 28.9 17 PF3D7_0503400 actin-depolymerizing factor 1 (ADF1) 3 28.7 9 PF3D7_0722400 GTP-binding protein, putative 6 28.5 29 ubiquitin-40S ribosomal protein S27a, PF3D7_1402500 putative 6 28.2 14 PF3D7_1331800 60S ribosomal protein L23, putative 3 28.1 14 PF3D7_1431700 60S ribosomal protein L14, putative 6 27.9 5 PF3D7_0918000 secreted acid phosphatase (GAP50) 8 27.8 10 PF3D7_0903700 alpha tubulin 1 8 27.6 22 conserved Plasmodium protein, unknown PF3D7_1459400 function 7 27.6 15 PF3D7_1351400 60S ribosomal protein L17, putative 5 27.6 15 T-complex protein 1 epsilon subunit, PF3D7_0320300 putative 13 27.5 28 1-acyl-sn-glycerol-3-phosphate PF3D7_1444300 acyltransferase, putative (LPAAT) 4 27.1 8 PF3D7_0210100.1 60S ribosomal protein L37ae, putative 3 27.1 4 PF3D7_1421200 40S ribosomal protein S25 (RPS25) 4 26.7 6 T-complex protein 1, gamma subunit, PF3D7_1229500 putative 12 26.6 35 conserved Plasmodium protein, unknown PF3D7_1457300 function 10 26.4 28 PF3D7_1441200 60S ribosomal protein L1, putative 7 26.3 6 PF3D7_0608700 chaperone, putative 11 26.2 25 PF3D7_0612900 nucleolar GTP-binding protein 1, putative 11 26.2 21 PF3D7_1222300 endoplasmin, putative (GRP94) 14 25.5 30 PF3D7_0627500 protein DJ-1 (DJ1) 3 25.4 4 PF3D7_0903200 ras-related protein RAB7 (RAB7) 4 25.2 9 PF3D7_0217800 40S ribosomal protein S26 (RPS26) 3 25.2 4 PF3D7_0930300;PF3 D7_1312000 merozoite surface protein 1 (MSP1) 31 24.6 54 signal recognition particle subunit SRP68, PF3D7_0621900 putative (SRP68) 12 24.5 21 conserved Plasmodium protein, unknown PF3D7_0513200 function 53 24.2 138 PF3D7_1309100 60S ribosomal protein L24, putative 5 24.1 8 PF3D7_0511800 inositol-3-phosphate synthase (INO1) 13 24 16 PF3D7_1445200 RNA helicase, putative 16 23.9 18 213

conserved Plasmodium protein, unknown PF3D7_1323900 function 7 23.7 13 conserved Plasmodium protein, unknown PF3D7_0423300 function 11 22.9 15 rRNA processing and telomere maintaining PF3D7_0925200 methyltransferase, putative 7 22.8 4 PF3D7_0710600 60S ribosomal protein L34 (RPL34) 4 22.7 30 knob-associated histidine-rich protein PF3D7_0202000 (KAHRP) 11 22.6 2 ADP/ATP transporter on adenylate PF3D7_1037300 translocase (ADT) 5 22.6 16 PF3D7_1252100 rhoptry neck protein 3 (RON3) 40 22.5 89 PF3D7_0812400 karyopherin alpha (KARalpha) 8 22.2 22 PF3D7_1238100 calcyclin binding protein, putative 4 21.5 11 PF3D7_0719600 60S ribosomal protein L11a, putative 4 21.4 8 PF3D7_0517300 pre-mRNA-splicing factor (SR1) 6 21.1 7 conserved Plasmodium membrane protein, PF3D7_1237700 unknown function 3 21 8 PF3D7_1124900 60S ribosomal protein L35, putative 3 21 4 S-adenosyl-L-homocysteine hydrolase PF3D7_0520900 (SAHH) 8 20.9 39 PF3D7_0614500 60S ribosomal protein L19 (RPL19) 5 20.9 9 26S proteasome regulatory subunit RPN11, PF3D7_1368100 putative (RPN11) 4 20.9 4 PF3D7_0706000 importin-7, putative 19 20.8 43 conserved Plasmodium protein (10b PF3D7_1021900 antigen), unknown function 38 20.4 100 PF3D7_1405900 RNA-binding protein, putative 18 20.4 28 26S protease regulatory subunit 10B, PF3D7_1306400 putative (RPT4) 6 20.4 15 PF3D7_0304400 60S ribosomal protein L44 (RPL44) 6 20.2 13 PF3D7_1407100 fibrillarin, putative (NOP1) 5 20.1 16 autophagy-related protein 18, putative PF3D7_1012900 (ATG18) 6 20 10 PF3D7_1108400 casein kinase 2, alpha subunit (CK2alpha) 5 20 26 PF3D7_1020700 histone acetyltransferase, putative 19 19.9 36 eukaryotic translation initiation factor 2 PF3D7_1010600 beta subunit, putative 3 19.8 12 conserved Plasmodium membrane protein, PF3D7_1409400 unknown function 4 19.1 11 PF3D7_1021500 RNA helicase, putative 10 19 16 eukaryotic translation initiation factor 2 PF3D7_1410600 gamma subunit, putative 4 18.7 18 PF3D7_1472200 histone deacetylase, putative (HDA1) 38 18.6 139 PF3D7_1008800 nucleolar protein 5, putative (NOP5) 8 18.1 23 PF3D7_0709700;PF3 D7_0102400 lysophospholipase, putative 4 17.9 16 PF3D7_0827900 protein disulfide isomerase (PDI8) 5 17.6 12 214

6-phosphogluconate dehydrogenase, PF3D7_1454700 decarboxylating, putative 6 17.3 16 PF3D7_1104400 conserved protein, unknown function 6 17 17 conserved Plasmodium protein, unknown PF3D7_0813300 function 4 16.8 18 PF3D7_1332900 isoleucine--tRNA ligase, putative 16 16.7 46 PF3D7_0214000 T-complex protein 1, putative 5 16.6 22 PF3D7_1205900 conserved protein, unknown function 13 16.5 21 eukaryotic translation initiation factor 3 PF3D7_1206200 subunit 8, putative 15 16.4 40 26S protease regulatory subunit 7, putative PF3D7_1311500 (RPT1) 4 16.2 23 26S protease regulatory subunit 8, putative PF3D7_1248900 (RPT6) 4 16.1 5 PF3D7_1134000 heat shock protein 70 (HSP70-3) 8 16 19 conserved Plasmodium protein, unknown PF3D7_1306200 function 5 15.9 8 PF3D7_1134100;PF3 D7_0710000 protein disulfide isomerase (PDI-11) 5 15.4 11 PF3D7_1108700 heat shock protein DnaJ homologue Pfj2 5 15.2 22 conserved Plasmodium protein, unknown PF3D7_1138700 function 20 15 41 PF3D7_1241700 replication factor C subunit 4, putative 3 14.9 6 haloacid dehalogenase-like hydrolase PF3D7_1033400 (HAD1) 3 14.6 13 conserved Plasmodium protein, unknown PF3D7_0932800 function 13 14.5 29 PF3D7_0306800 T-complex protein beta subunit, putative 4 14.1 58 protein transport protein SEC61 subunit PF3D7_1346100 alpha (SEC61) 4 14 8 eukaryotic translation initiation factor 3 PF3D7_1212700 subunit 10, putative 14 13.9 40 Plasmodium exported protein (PHISTb), PF3D7_0401800 unknown function (PfD80) 9 13.9 8 PF3D7_0312400 glycogen synthase kinase 3 (GSK3) 3 13.9 9 PF3D7_0815200 importin beta, putative 8 13.8 22 ribonucleoside-diphosphate reductase, PF3D7_1437200 large subunit, putative 8 13.8 20 mature parasite-infected erythrocyte surface antigen,erythrocyte membrane PF3D7_0500800 protein 2 (MESA) 11 13.7 25 PF3D7_0525100 acyl-CoA synthetase (ACS10) 7 13.7 18 PF3D7_1318800 translocation protein SEC63 (SEC63) 6 13.7 12 PF3D7_1218300 AP-3 complex subunit mu, putative 6 13.5 4 golgi organization and biogenesis factor, PF3D7_0418200 putative 4 13.5 12 PF3D7_1308900 mRNA-decapping enzyme 2, putative (DCP2) 14 13.4 15 PF3D7_0915400 6-phosphofructokinase (PFK9) 10 13.3 75 PF3D7_1419700 conserved Plasmodium protein, unknown 4 13 22 215

function proliferation-associated protein 2g4, PF3D7_1428300 putative 5 12.7 18 PF3D7_1437900 HSP40, subfamily A, putative 3 12.7 21 PF3D7_0102900 aspartate--tRNA ligase 5 12.6 17 PF3D7_1368200 RNAse L inhibitor protein, putative 5 12.6 18 PF3D7_1471100 exported protein 2 (EXP2) 3 12.5 6 PF3D7_1145100 coatomer subunit gamma, putative 7 12.3 15 rab specific GDP dissociation inhibitor PF3D7_1242800 (rabGDI) 3 12.2 12 ATP-dependent RNA helicase DBP10, PF3D7_0827000 putative (DBP10) 10 12.1 20 PF3D7_1228600 merozoite surface protein 9 (MSP9) 6 12 10 26S proteasome regulatory subunit RPN6 PF3D7_1402300 (RPN6) 4 12 8 PF3D7_1213800 proline--tRNA ligase (PRS) 6 11.9 26 conserved Plasmodium protein, unknown PF3D7_0111800 function 5 11.9 11 signal recognition particle receptor, beta PF3D7_1246800 subunit (SRPR-beta) 3 11.9 8 conserved Plasmodium protein, unknown PF3D7_1361800 function 17 11.8 84 ATP-dependent RNA helicase DDX5, PF3D7_1445900 putative (DDX5) 5 11.8 19 PF3D7_1132200 TCP-1/cpn60 chaperonin family, putative 5 11.8 10 PF3D7_1235600 serine hydroxymethyltransferase (SHMT) 5 11.8 5 PF3D7_0106300 calcium-transporting ATPase (ATP6) 9 11.4 29 conserved Plasmodium protein, unknown PF3D7_0821000 function 5 11.3 13 isocitrate dehydrogenase (NADP), PF3D7_1345700 mitochondrial precursor (IDH) 3 11.3 7 PF3D7_0817700 rhoptry neck protein 5 (RON5) 10 11.2 46 PF3D7_1401800 choline kinase (CK) 4 11.1 13 U3/U14 snoRNA-associated small subunit PF3D7_1109400 rRNA processing protein, putative 3 11 7 conserved Plasmodium protein, unknown PF3D7_0108300 function 20 10.8 59 PF3D7_0716600 cysteine desulfurase (SufS) 4 10.8 23 mRNA binding Pumilio-homology domain PF3D7_0621300 protein, putative 7 10.6 9 PF3D7_1342600 myosin A (MyoA) 5 10.5 28 PF3D7_1203000 origin recognition complex subunit 1 (ORC1) 8 10.4 8 PF3D7_0320800 ATP-dependent RNA helicase DDX6 (DOZI) 3 9.9 7 PF3D7_0212900 Leu/Phe-tRNA protein transferase, putative 3 9.9 7 PF3D7_0113000 glutamic acid-rich protein (GARP) 6 9.8 32 26S proteasome regulatory subunit RPN3, PF3D7_1338100 putative (RPN3) 3 9.7 8 PF3D7_1354300 large subunit rRNA methyltransferase, 10 9.4 27 216

putative 26S proteasome regulatory subunit p55, PF3D7_1017900 putative (RPN5) 3 9.4 8 PF3D7_0617100 AP-2 complex subunit alpha, putative 10 9.3 14 FACT complex subunit SSRP1, putative PF3D7_1441400 (FACT-S) 3 9.3 15 PF3D7_1218600 arginine--tRNA ligase, putative 3 9.2 13 PF3D7_0910100 Ran-binding protein, putative 8 8.9 18 eukaryotic translation initiation factor, PF3D7_0517700 putative 5 8.9 11 inorganic anion exchanger, inorganic anion PF3D7_1471200 antiporter (SulP) 4 8.9 7 polyadenylate-binding protein, putative PF3D7_1224300 (PABP) 7 8.8 30 small subunit rRNA processing factor, PF3D7_1225500 putative 3 8.8 8 conserved Plasmodium protein, unknown PF3D7_1026000 function 3 8.8 10 PF3D7_1329100 myosin C (MyoC) 15 8.6 26 PF3D7_1436000 glucose-6-phosphate isomerase (GPI) 4 8.6 7 PF3D7_1419100 ATP-dependent RNA helicase, putative 6 8.5 11 PF3D7_1325100 phosphoribosylpyrophosphate synthetase 3 8.5 3 phosphopantetheine adenylyltransferase, PF3D7_0704700 putative (PPAT) 9 8.4 8 conserved Plasmodium protein, unknown PF3D7_1356100 function 5 8.3 18 conserved Plasmodium protein, unknown PF3D7_1411500 function 5 8.2 24 PF3D7_1417800;PF3 DNA replication licensing factor MCM2 D7_0525600 (MCM2) 5 8.2 22 PF3D7_1022400 serine/arginine-rich splicing factor 4 (SRSF4) 4 8.2 12 PF3D7_1145400 dynamin-like protein (DYN1) 4 8.1 22 PF3D7_1311800 M1-family alanyl aminopeptidase (M1AAP) 6 7.4 21 conserved Plasmodium protein, unknown PF3D7_1246300 function 4 7.4 10 PF3D7_1238800 acyl-CoA synthetase (ACS11) 4 7.2 13 PF3D7_0708800 heat shock protein 110 (HSP110c) 3 7.2 36 calcium-dependent protein kinase 1 PF3D7_0217500 (CDPK1) 3 7.1 9 PF3D7_1232100 60 kDa chaperonin (CPN60) 3 7 1 PF3D7_0801800 mannose-6-phosphate isomerase, putative 5 6.9 32 DEAD/DEAH box ATP-dependent RNA PF3D7_1251500 helicase, putative 3 6.8 4 conserved Plasmodium protein, unknown PF3D7_1141300 function 6 6.7 16 cell division cycle protein 48 homologue, PF3D7_0619400 putative 3 6.6 15 eukaryotic translation initation factor 4 PF3D7_1312900 gamma, putative (EIF4G) 6 6.5 15 217

PF3D7_0106100 vacuolar ATP synthase subunit c, putative 3 6.5 7 conserved Plasmodium protein, unknown PF3D7_1248700 function 9 6.4 25 conserved Plasmodium protein, unknown PF3D7_0418300 function 5 6.4 6 PF3D7_1446200 M17 leucyl aminopeptidase (LAP) 3 6.4 6 glideosome-associated protein 40, putative PF3D7_0515700 (GAP40) 3 6.1 3 conserved Plasmodium protein, unknown PF3D7_1359600 function 8 6 66 PF3D7_0523000 multidrug resistance protein (MDR1) 5 6 25 PF3D7_0416400 histone acetyltransferase, putative (HAT1) 4 6 15 snoRNA-associated small subunit rRNA PF3D7_1217200 processing protein, putative 4 6 8 PF3D7_1015600 heat shock protein 60 (HSP60) 3 6 0 PF3D7_1452000 rhoptry neck protein 2 (RON2) 10 5.9 60 PF3D7_1219100 clathrin heavy chain, putative 8 5.9 43 DNA mismatch repair protein MSH2, PF3D7_1427500 putative (MSH2-1) 4 5.9 16 ubiquitin carboxyl-terminal hydrolase 1, PF3D7_0104300 putative (UBP1) 13 5.8 30 FACT complex subunit SPT16, putative PF3D7_0517400 (FACT-L) 5 5.8 19 lysine-specific histone demethylase 1, PF3D7_1211600 putative (LSD1) 12 5.4 79 conserved Plasmodium membrane protein, PF3D7_1419400 unknown function 7 5.4 30 eukaryotic translation initiation factor 3, PF3D7_0528200 subunit 6, putative 3 5.4 13 conserved Plasmodium protein, unknown PF3D7_0824900 function 3 5.4 5 PF3D7_0302900 exportin-1, putative 4 5.2 29 PF3D7_0528100 AP-1 complex subunit beta, putative 3 5.2 11 PF3D7_1436600 cGMP-dependent protein kinase (PKG) 3 5.2 8 PF3D7_1445100 histidine--tRNA ligase, putative 5 5 14

218

Appendix IV: Proteins of interest found similar between MyoB-BirA* and MTIPB-BirA* mass spectrometry

Seq Seq Peptides Peptides Peptides cov. cov. Seq cov Gene ID Fasta Header MyoB MTIPB 3D7 MyoB MTIPB 3D7 [%] [%] [%] conserved Plasmodium protein, unknown PF3D7_0710200 function 105 39 0 40.8 17.4 0

PF3D7_0503600 myosin B 65 7 0 73.8 12.6 0 conserved Plasmodium protein, unknown PF3D7_1014900 function 58 16 0 27.6 8.2 0

myosin light PF3D7_1118700 chain B 38 64 0 57.5 72.4 0 DNA replication licensing factor MCM3, PF3D7_0527000 putative 25 19 1 36.1 29.8 1.8 Conserved unknown PF3D7_1138000 function 22 54 0 11.1 20.5 0

PF3D7_0914100 cytoplasm 16 19 2 15.9 16.8 1.5 Conserved unknown PF3D7_1207000 function 16 23 0 7.2 10.7 0 regulator of chromosome condensation, PF3D7_0919900 putative 15 9 0 7.6 5.3 0 219

conserved Plasmodium protein, unknown PF3D7_0508900 function 14 11 0 6.8 5.5 0 MCM4 complex, PF3D7_1317100 nucleus 14 13 2 22.1 17.5 3.7 conserved Plasmodium protein, unknown PF3D7_0630600 function 11 12 1 15.5 16.4 2 Conserved unknown PF3D7_1227700 function 10 17 0 18.3 24 0 MCM6 complex, cytoplasm, PF3D7_1355100 nucleus 10 9 0 14.3 14 0

PF3D7_1446600 centrin-2 8 4 0 73.2 56.5 0

PF3D7_1412500 actin filament 7 6 2 31.9 29 19.7 dynamin-like PF3D7_1037500 protein 7 3 0 18.6 7.1 0 MCM7 complex, cytoplasm, PF3D7_0705400 nucleus 7 11 2 12.1 17.5 2.9 gamma-tubulin complex, PF3D7_0803700 microtubule 5 6 1 15.5 17.9 2.2 conserved Plasmodium protein, unknown PF3D7_0408100 function 5 7 0 8.3 8 0 DNA replication licensing factor MCM5, PF3D7_1211700 putative 9 8 1 17.4 12.4 1.2