bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424754; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Analysis of a Mad2 homolog in Trypanosoma brucei provides

possible hints on the origin of the spindle checkpoint

Bungo Akiyoshi*

Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK

*For correspondence: [email protected]

Abstract

The spindle checkpoint is a surveillance mechanism that ensures accurate nuclear DNA segregation in

eukaryotes. It does so by delaying the onset of anaphase until all kinetochores have established proper

attachments to spindle microtubules. The evolutionary origin of the spindle checkpoint remains

unclear. The flagellated kinetoplastid parasite Trypanosoma brucei has a nucleus that contains the

nuclear genome and a kinetoplast that contains the mitochondrial genome. The kinetoplast is

physically linked to the basal body of the flagellum and its segregation is driven by the movement of

basal bodies. While there is no strong evidence that T. brucei possesses a functional spindle

checkpoint, it has been suggested that initiation of cytokinesis may be linked to the completion of

kinetoplast segregation or basal body separation in this organism. Interestingly, the only identifiable

spindle checkpoint component TbMad2 localizes at the basal body area. Here, I report identification

of that co-purified with TbMad2. One , which I propose to call TbMBP65, localizes at

the basal body area and has a putative Mad2-interacting motif. Interestingly, 26S proteasome subunits

also co-purified with TbMad2, raising a possibility that TbMad2 might regulate proteasome activity to

regulate or monitor the segregation of basal bodies. I speculate that such a function might represent a

prototype of the spindle checkpoint. I also show that TbAUK3, one of the three Aurora kinase

homologs in T. brucei, localizes at the basal body area from late G1 until the time of kinetoplast

separation. Immunoprecipitation of TbAUK3 identified an uncharacterized protein (termed

TbABP79) that has a similar localization pattern as TbAUK3. These findings provide a starting point

to reveal the function of TbMad2 and TbAUK3 as well as the origin of the spindle checkpoint.

1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424754; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Introduction

It is essential that cells transmit their genetic material and organelles accurately at each cell division.

Kinetochores are the macromolecular protein complexes that assemble onto centromeric DNA and

interact with spindle microtubules in eukaryotes (Musacchio and Desai, 2017). Accurate

segregation requires that kinetochores attach to microtubules emanating from opposite poles. The

spindle checkpoint is a surveillance system that ensures high fidelity segregation of nuclear DNA

(Murray, 2011). In response to defects in kinetochore-microtubule attachments, the spindle

checkpoint delays the onset of anaphase by inhibiting the multi-subunit ubiquitin ligase called the

anaphase-promoting complex/cyclosome (APC/C) (Musacchio and Salmon, 2007; London and

Biggins, 2014; Musacchio, 2015; Alfieri et al., 2017). Once all have achieved proper

attachments, the spindle checkpoint is satisfied, leading to the ubiquitylation of two key targets

(cyclin B and securin) by the APC/CCdc20. Ubiquitylated proteins are subsequently destroyed by the

26S proteasome, triggering chromosome segregation and the exit of mitosis. Spindle checkpoint

components include Mad1, Mad2, Mad3 (BubR1), Mps1, Bub1 and Bub3. These proteins are widely

conserved among eukaryotes and it is therefore thought that the last eukaryotic common ancestor had

a spindle checkpoint (Vleugel et al., 2012; van Hooff et al., 2017; Tromer et al., 2019; Kops et al.,

2020). A key player Mad2 is recruited to the kinetochore by direct interaction with Mad1, which

promotes conformational conversion of Mad2 so that it that can interact with Cdc20 (De Antoni et al.,

2005). Mad1 and Cdc20 have similar Mad2-interacting motifs (K/R-Φ-Φ-x-x-x-x-x-P, where Φ is an

aliphatic residue and x is any residue) (Luo et al., 2002; Sironi et al., 2002). Although there have been

extensive studies in select model eukaryotes that all belong to Opisthokonta (e.g. yeasts, worms, flies,

and human), very little is known about the spindle checkpoint or cell cycle regulation in

evolutionarily divergent eukaryotes (Kaczanowski et al., 1985; Morrissette and Sibley, 2002; Vicente

and Cande, 2014; Komaki and Schnittger, 2016; Markova et al., 2016).

While segregation of nuclear DNA depends on spindle microtubules in all studied eukaryotes,

the mechanism for mitochondrial DNA transmission varies among eukaryotes. Animals have a high

copy number of mitochondria and their transmission is thought to occur randomly (Westermann,

2010). In contrast, a single mitochondrion is present in some unicellular eukaryotes, such as

2 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424754; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Trypanosoma brucei (the kinetoplastid parasite that causes human African trypanosomiasis,

Discobids), Plasmodium falciparum (the parasite that causes malaria, Alveolates), and

Cyanidioschyzon merolae (red algae, Archaeplastids) (Vickerman, 1962; Itoh et al., 1997; Okamoto et

al., 2009; Walker et al., 2011; Keeling and Burki, 2019). T. brucei has a kinetoplast that contains

mitochondrial DNA (kDNA) and a nucleus that contains nuclear DNA (Figure 1). Kinetoplasts

segregate prior to the nuclear division, followed by cytokinesis (Woodward and Gull, 1990; Siegel et

al., 2008). It is essential that cells duplicate and segregate these single-copy organelles accurately at

each cell division cycle. The kDNA is physically attached to the basal body of the flagellum via the

tripartite attachment complex, and its segregation is driven by the separation of basal bodies/flagella

(Robinson and Gull, 1991; Ogbadoyi et al., 2003; Schneider and Ochsenreiter, 2018). T. brucei does

not break down its nuclear envelope (closed mitosis), and an intranuclear mitotic spindle forms in the

nucleus during mitosis (Vickerman and Preston, 1970; Ogbadoyi et al., 2000). It is thought that T.

brucei does not have a canonical spindle checkpoint because cells are unable to halt their cell cycle in

response to spindle defects and undergo cytokinesis without a noticeable delay despite a lack of

nuclear division (Robinson et al., 1995; Ploubidou et al., 1999; Hayashi and Akiyoshi, 2018), which is

consistent with the apparent absence of all the spindle checkpoint components mentioned above

(except for a Mad2 homolog called TbMad2, see below) as well as the absence of the well-conserved

Mad2-interacting motif in TbCdc20 (Akiyoshi and Gull, 2013). Cyclin B (called CYC6 in T. brucei)

localizes at the basal body area in S phase and appears in the nucleus in G2 phase until it disappears at

the onset of anaphase (Hayashi and Akiyoshi, 2018). Forced stabilization of cyclin B in the nucleus

causes the nucleus to arrest in a metaphase-like state without preventing cytokinesis, suggesting that

trypanosomes have an ability to regulate the timing of nuclear division by modulating the cyclin B

protein level without a canonical spindle checkpoint.

It has been proposed that kinetoplastids might be among the earliest-branching eukaryotes

based on various distinct features, such as the unique cytochrome c biogenesis machinery and

unconventional kinetochore components (Allen et al., 2008; Cavalier-Smith, 2010; Akiyoshi, 2016).

Understanding the biology of T. brucei could therefore provide insights into the origin of the spindle

checkpoint and/or regulatory mechanisms for organelle segregation. I previously showed in procyclic

3 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424754; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

(insect) form trypanosome cells that TbMad2 localizes at the basal body area throughout the cell cycle

(Akiyoshi and Gull, 2013), which was verified in other studies (Dean et al., 2017; Zhou et al., 2019).

No kinetochore signal was observed even when spindle microtubules were disrupted (Akiyoshi and

Gull, 2013; Zhou et al., 2019), suggesting that TbMad2 unlikely plays a role in the canonical spindle

checkpoint. In this study, I immunoprecipitated TbMad2 and identified another protein that localizes

at the basal body area. I also identified two proteins that transiently localize at the basal body area.

Results and Speculations

Identification of a TbMad2-binding protein, TbMBP65

Mad2 belongs to the HORMA domain family, which includes Hop1, Rev7, and Mad2 (Aravind and

Koonin, 1998; Rosenberg and Corbett, 2015; Tromer et al., 2019; Ye et al., 2020). Multiple sequence

alignment of Mad2 proteins from various eukaryotes clearly shows that TbMad2 has high higher

sequence similarity to Mad2 than to other HORMA domain containing proteins (Figure S1 and data

not shown). Furthermore, many residues that are important for Mad2’s functions (Luo et al., 2000,

2002; Sironi et al., 2002; De Antoni et al., 2005; Mapelli et al., 2007) are conserved in TbMad2

(Figure S1). However, TbMad2 localizes at the basal body region (Akiyoshi and Gull, 2013; Dean et

al., 2017; Zhou et al., 2019). To identify its interaction partners, I immunoprecipitated the YFP-tagged

TbMad2 protein from procyclic cells and performed mass spectrometry (Table S1). This led to the

identification of Tb927.6.1220 (Figure 2A), which was recently identified in the BioID of TbSpef1

(Sperm flagellar protein 1: also known as CaLponin-homology And Microtubule-associated Protein

(CLAMP) or TbCMF18) (Chan et al., 2005; Dougherty et al., 2005; Broadhead et al., 2006; Baron et

al., 2007; Gheiratmand et al., 2013; Dong et al., 2020). TbSpef1 localizes at the microtubule quartet

that originates from the basal body and extends toward the anterior end of the cell. As previously

shown (Dong et al., 2020), Tb927.6.1220 localizes at the basal body area throughout the cell cycle

(Figure 2B). Some cells additionally had signals near the anterior tip (Figure 2B, arrowhead).

Interestingly, I found a putative Mad2-interacting motif (Figure 2C), suggesting that Tb927.6.1220

may directly interact with TbMad2. I therefore propose to call this protein TbMBP65 for TbMad2-

Binding Protein 65 kDa. Its putative homologs were identified in kinetoplastids, diplonemids, and

4 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424754; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

euglenids (Figure S2), but not outside of Euglenozoan species. The putative Mad2-interacting motif

of TbMBP65 homologs is present in most kinetoplastids but is largely absent in diplonemids and

euglenids. It is possible that TbMBP65 is a receptor at the basal body area that recruits TbMad2,

similarly to how Mad1 recruits Mad2 onto kinetochores in other eukaryotes. Alternatively, TbMBP65

might play an effector role, similarly to the function of Cdc20 in the canonical spindle checkpoint.

To my surprise, TbMad2 also co-purified with RPT1/2/3/5 and RPN1/11 (Figure 2A), which

are subunits of the 19S regulatory particle of the 26S proteasome (Li and Wang, 2002; Bard et al.,

2018). These proteins were not detected in the immunoprecipitate of any other protein I purified using

the same method (Table S1 and data not shown), suggesting that the observed co-purification is likely

significant. Although totally speculative, it is interesting to discuss a possibility that TbMad2 might

regulate the activity of proteasomes, which would differ from the canonical spindle checkpoint that

more indirectly prevents substrate degradation via inhibition of the APC/C ubiquitin ligase (Figure

2D). Given that kinetoplastids might be one of the earliest-branching eukaryotes (Cavalier-Smith,

2010; Akiyoshi, 2016), the interaction between TbMad2 and proteasome regulatory subunits might

represent an ancient mode for the regulation of the ubiquitin/proteasome-dependent substrate

degradation system. It is noteworthy that another divergent eukaryote Giardia intestinalis

(Metamonads) lacks APC/C components but has a Mad2 homolog (Eme et al., 2011; Gourguechon et

al., 2013; Vicente and Cande, 2014; Markova et al., 2016). It will be interesting to examine whether

GiMad2 co-purifies with proteasome subunits.

It is thought that basal bodies/flagella (centrioles/cilia) were already present in the last

eukaryotic common ancestor (Carvalho-Santos et al., 2011), so it is conceivable that transmission of

these organelles was critical for survival and that early eukaryotes might have had a checkpoint to

monitor their proper inheritance. Given that TbMad2 localizes at the basal body area, it might regulate

and/or monitor the duplication or segregation of basal bodies (or associated structures) in T. brucei,

which possibly represents a prototype of the spindle checkpoint.

TbAUK3 and its putative binding protein TbABP79 transiently localize at the basal body area

5 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424754; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Aurora kinases play various key roles in eukaryotes, including regulation of mitosis and flagellar/cilia

disassembly (Pan et al., 2004; Marumoto et al., 2005; Pugacheva et al., 2007; Inoko et al., 2012;

Hochegger et al., 2013; Carmena et al., 2015; Korobeynikov et al., 2017; Bertolin and Tramier, 2020).

Trypanosoma brucei has three Aurora kinases, TbAUK1, TbAUK2, and TbAUK3, among which

TbAUK1 has a similar localization pattern as the Aurora B kinase in human (Li et al., 2008). As part

of an effort towards identifying more mitotic proteins, I examined the localization of TbAUK2 and

TbAUK3 (Tu et al., 2006; Jones et al., 2014; Fernandez-Cortes et al., 2017; Stortz et al., 2017)

(Figure S3). TbAUK2 had a diffuse nuclear signal (data not shown), while TbAUK3 localized at the

basal body area in a cell cycle dependent manner (Figure 3). Interestingly, TbAUK3 signal was not

found in all 1K1N cells, suggesting that TbAUK3 starts to localize in late G1. In 1K*1N cells, there

were multiple signals, which may reflect its localization at duplicated basal bodes or associated

structures. Then the signals disappeared once the kDNA appeared as two distinct dots.

I then performed its immunoprecipitation and mass spectrometry, identifying Tb927.11.340

as a hit (Figure 4A and Table S1). This protein had a largely similar localization pattern as TbAUK3

(Figure 4B), so I propose to call it TbABP79 for TbAUK3-Binding Protein 79 kDa. No obvious

homolog was found outside of kinetoplastids (Figure S4). The unique localization pattern of TbAUK3

and TbABP79 raises a possibility that these proteins might be involved in the biogenesis or separation

of basal bodies or associated structures. Alternatively, they might monitor the segregation status of

basal bodies or kinetoplasts.

Conclusions

In this manuscript, I reported several proteins that localize near basal bodies in T. brucei. I hope that

this preprint will encourage some researchers to study these proteins in more detail and reveal their

function in the future. It will be important to determine their exact location, for example by comparing

with components of basal bodies, the microtubule quartet, hook complex components, and flagellar

pocket collar (Lacomble et al., 2009; Esson et al., 2012; Albisetti et al., 2017; Halliday et al., 2019;

Dong et al., 2020). It is also critical to reveal their knockdown or knockout phenotype. Some spindle

checkpoint mutants show defects only in the presence of microtubule drugs (Hoyt et al., 1991; Li and

6 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424754; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Murray, 1991), so it might be necessary to identify proper stress conditions to detect a phenotype. I

emphasize that there is currently no evidence to suggest that TbMad2 and TbAUK3 have similar

functions. However, there are relationships among the ubiquitin-proteasome system, Aurora kinase,

and basal body/centriole/cilia (Kasahara et al., 2014; Izawa et al., 2015; Shearer and Saunders, 2016),

so it will be worth examining whether inhibition of TbMad2 or proteasome activity affects TbAUK3

and vice versa. Interestingly, treatment of procyclic trypanosome cells with a proteasome inhibitor

lactacystin caused accumulation of cells with one kinetoplast and one nucleus with replicated DNA

(Mutomba et al., 1997). Although lack of nuclear division can be explained by stabilization of cyclin

B (Hayashi and Akiyoshi, 2018), why did these cells (apparently) have one kinetoplast and fail to

divide? Because kinetoplast segregation depends on basal body separation (Robinson and Gull, 1991),

one possible explanation is that proteasome activity may be required for the separation of basal bodies.

In this scenario, the lack of cytokinesis could be explained by the hypothesis that initiation of

cytokinesis may be linked to the completion of the kinetoplast segregation or basal body segregation

in trypanosomes (Ploubidou et al., 1999). It will be interesting to revisit the phenotype of lactacystin-

treated cells by examining the status of basal bodies, other associated structures, and cytokinesis.

7 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424754; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Materials and Methods

Trypanosome cells and plasmids and microscopy

Trypanosome cell lines, plasmids, and primers used in this study are listed in Table S2, S3, and S4.

All cell lines used in this study were derived from T. brucei SmOxP927 procyclic form cells (TREU

927/4 expressing T7 RNA polymerase and the tetracycline repressor to allow inducible expression)

(Poon et al., 2012). Cells were grown at 28˚C in SDM-79 medium supplemented with 10% (v/v) heat-

inactivated fetal calf serum (Brun and Schönenberger, 1979). Endogenous YFP tagging constructs

were made using the pEnT5-Y vector (Kelly et al., 2007) with the DNA Strider software (Marck,

1988). Transfected cells were selected by the addition of 25 µg/ml hygromycin. Fluorescence

microscopy was performed as described previously (Akiyoshi and Gull, 2014).

Immunoprecipitation and mass spectrometry

Immunoprecipitation of YFP-TbMad2 and YFP-TbAUK3 was carried out using anti-GFP antibodies

as described previously (Akiyoshi and Gull, 2014) with the following modifications. 400 ml cultures

of asynchronously growing cells were harvested at ~1.2 x 107 cells/ml. Cells were pelleted by

centrifugation (900 g, 15 min), washed once with PBS, resuspended in 0.5 ml of BH0.15 (25 mM

HEPES pH 8.0, 2 mM MgCl2, 0.1 mM EDTA, 0.5 mM EGTA, 1% NP-40, 150 mM KCl, and 15%

glycerol) with protease inhibitors (20 µg/ml each of leupeptin, pepstatin, and E-64 as well as 0.2 mM

PMSF) and phosphatase inhibitors (1 mM sodium pyrophosphate, 2 mM Na-beta-glycerophosphate,

0.1 mM Na3VO4, 5 mM NaF, and 200 nM microcystin-LR), and then frozen in small drops into liquid

nitrogen in a mortar. Cells were ground with a pestle with liquid nitrogen, and the cell power was

transferred into a 50 ml tube. When the samples started to melt, 1 ml of BH0.15 with inhibitors was

added, followed by 25 min incubation to allow lysis on ice. The samples were then centrifuged at

14,000 rpm for 30 min at 4 °C. The supernatant was transferred into a new tube and was mixed with

mouse monoclonal anti-GFP antibodies (11814460001, Roche) that had been pre-conjugated with

Protein-G Dynabeads with dimethyl pimelimidate using a protocol described in (Unnikrishnan et al.,

2012). Subsequent steps and mass spectrometry were performed essentially as described (Akiyoshi

8 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424754; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

and Gull, 2014), and peptides were identified with MASCOT (Matrix Science). Proteins identified

with at least two peptides were considered as significant and shown in Table S1.

Sequence analysis

Protein sequences and accession numbers for proteins used in this study were retrieved from TriTryp

database (Aslett et al., 2010), Wellcome Sanger Institute (https://www.sanger.ac.uk/), UniProt

(UniProt Consortium, 2019), or published studies (O’Neill et al., 2015; Butenko et al., 2020).

Homology searches were done using BLAST in TriTryp (Aslett et al., 2010), HMMER web server

(Potter et al., 2018), or hmmsearch using manually prepared HMM profiles (HMMER version 3.0)

(Eddy, 1998). Multiple sequence alignment was performed with MAFFT (L-INS-i method, version 7)

(Katoh et al., 2019) and visualized with the Clustalx coloring scheme in Jalview (version 2.11)

(Waterhouse et al., 2009). The HMM logo for the Mad2-interacting motif in Mad1 was created with

skylign (Wheeler et al., 2014) based on Mad1 homologs from various eukaryotes (van Hooff et al.,

2017). Coiled coils were predicted using COILS program (Lupas et al., 1991).

Acknowledgments

I thank Professor Keith Gull for his support. I also thank the Advanced Proteomics Facility, Benjamin

Thomas, and Gabriela Ridlova for mass spectrometry analysis. Experiments reported in this

manuscript were carried out when I was supported by postdoctoral fellowships from the EMBO and

Human Frontier Science Program. I am currently supported by a Wellcome Trust Senior Research

Fellowship (grant no. 210622/Z/18/Z) and the European Molecular Biology Organization Young

Investigator Program. I declare that no competing interests exist.

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A Nuclear DNA Basal body Flagellum 1K1N kDNA

B New flagellum New basal body 1K*1N Basal body segregation drives kinetoplast DNA segregation (Nucleus is in S phase) C

2K1N

D Nuclear DNA division

E 2K2N

F Cytokinetic furrow ingression Cytokinesis

G

Formation of two daughter cells

Figure 1.Figure Diagram 1. of Diagram the cell division of the cycle cell indivision Trypanosoma cycle brucei in Trypanosoma procyclic (insect) brucei form cells (A) G1 cells possess a single kinetoplastprocyclic and nucleus (insect) (termed form 1K1N) cells as well as an attached flagellum. (B) As the cell cycle progresses, a new basal body forms and nucleates a new flagellum. The nucleus is still in S phase when kinetoplast DNA (kDNA) has an elongated morphology (termed 1K*1N). (C) Segregation of basal bodies leads to the separation of attached kinetoplast (termed 2K1N). (D) Cells enter nuclear M phase, and chromosome segregation occurs. (E) Nuclear division is complete (termed 2K2N). (F) Cleavage furrow ingression occurs between the two flagella. (G) At the end of the cell cycle, two daughter cells are formed, and each cell inherits a single kinetoplast, nucleus, basal body and flagellum. This figure was reproduced and modified from (Akiyoshi and Gull, 2013) under CC-BY.

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A C Putative Mad2-interacting motif in TbMBP65

T.brucei_Tb927.6.1220 35 THRVM SPCRRPPV Proteins co-purified Mascot Number of T.cruzi_TcCLB.507603.60 33 NYRVLAPCMRPP- Coverage T.rangeli_TRSC58_05191 33 RFRVLAPCMRPP- with YFP-TbMad2 score peptides T.grayi_DQ04_01761100 32 NYRVLAPCKRPP- T.theileri_TM35_000321090 28 QYRVLAPTRR--- Tb927.6.1220 (TbMBP65) 559 44.2% 22 T.vivax_TvY486_0600730 92 TFRVMSPCRRPPG L.mexicana_LmxM.12.0070 78 AFRVWAPTQHPP- TbMAD2 551 41.8% 11 E.monterogeii_EMOLV88_120005700 34 T L R V W A P IQ H P P - RPT2 194 19.2% 5 L.seymouri_Lsey_0135_0090 36 TLRVWAPIRHPP- C.fasciculata_CFAC1_010006300 43 AFRVWAPAQHPP- RPT5 138 16.2% 3 B.ayalai_Baya_067_0260 32 HYRVWAPVNRPP- P.confusum_PCON_0057390 41 HFRVLAPSKRPP- RPT3 76 23.8% 4 Prokinetoplastina_PhM_4_TR15442 31 GVRVLAPK IKAAE

RPN1 75 14.3% 5 1 2 3 4 5 6 7 8 9 10 11 12 13 RPT1 60 13.7% 2 RPN11 53 11.8% 2 K T V H E L RD MKN AA K I EKNS T S QF L VQ R L DV E L P V S GK N L P SMT T Y Y ER V H FM Y CT R CWRYH HPY Mad2-interacting motif in Mad1 B YFP-TbMBP65 YFP-TbMBP65 YFP-TbMBP65 DAPI Phase contrast DAPI Phase contrast

G1 (1K1N) KN

S (1K*1N)

G2/ prometa (2K1N) Near anterior tip

Anaphase (2K2N)

D Speculative model Canonical spindle checkpoint TbMad2

Cdc20 APC/C Proteasome

Substrates Ubiquitylated substrates Degraded substrates

Figure 2. Identification of a putative TbMad2-binding protein, TbMBP65 Figure 2. Identification of a putative TbMad2-binding protein, TbMBP65 (A) Summary of proteins detected in the immunoprecipitate of YFP-TbMad2 by mass spectrometry. See Table S1 for all proteins identified by mass spectrometry. (B) YFP-TbMBP65 localizes near basal bodies throughout the cell cycle. Note that some cells additionally have signals near the anterior tip (arrowhead). One allele of TbMBP65 was endogenously tagged at the N-terminus with YFP. Cells were fixed with 4% formaldehyde and stained with DAPI. Scale bar, 10 µm. (C) Alignment of TbMBP65 homologs in kinetoplastids reveals a putative Mad2-interacting motif. Logos of the Mad2-interacting motif in Mad1 proteins are shown below. (D) A highly speculative hypothesis that TbMad2 might negatively regulate proteasome activity, which would differ from the canonical spindle checkpoint that regulate APC/CCdc20 activity.

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YFP-TbAUK3 YFP-TbAUK3 DAPI DAPI Phase contrast

G1 (1K1N)

S (1K*1N)

G2/ prometa (2K1N)

Anaphase (2K2N)

Figure 3. TbAUK3 localizes transiently at the basal body area Figure 3. TbAUK3 localizes transiently at the basal body area One allele of TbAUK3 was endogenously tagged at the N-terminus with YFP. Cells were fixed with 4% formaldehyde and stained with DAPI. Scale bar, 10 µm.

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A Proteins co-purified Mascot Number of Coverage with YFP-TbAUK3 score peptides

Tb927.11.340 (TbABP79) 309 24.3 10 AUK3 72 13.2 3

B YFP-TbABP79 YFP-TbABP79 DAPI DAPI Phase contrast

G1 (1K1N)

S (1K*1N)

G2/ prometa (2K1N)

Anaphase (2K2N)

Figure 4. A putative TbAUK3-binding protein, TbABP79, Figure 4. A putative TbAUK3also-binding localizes protein, transiently TbABP79, at the basal also body localizes area transiently at the basal body area (A) Summary of proteins detected in the immunoprecipitate of YFP-TbAUK3 by mass spectrometry. See Table S1 for all proteins identified by mass spectrometry. (B) One allele of TbABP79 was endogenously tagged at the N-terminus with YFP. Cells were fixed with 4% formaldehyde and stained with DAPI. Scale bar, 10 µm.

20 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424754; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Supplemental Materials

Supplemental Figures

Tbrucei_TbMAD2 1 MAERLQTITF-RSSVSTITEFLGFAIYCVLYQRGVYPSDSF------QQVTRYGVQ--LMVSVDE 56 T.cruzi_TcCLB.510091.40 1 MSQAVQTITF-KGSVAMVTEFLGFAIYSILYQRGVYPQ EVF------EQVKRYG IP--LMISTDS 56 T.vivax_TvY486_0301060 1 MSSAEQCLTF-RSSVSTVAELLGFAIYSILYQRGVFPCESF------SQVTRYGMP--LMVSSDA 56 L.mexicana_LmxM.25.1670 1 MTQTTHAISL-TGSVATVTEYLGFAINNILYQRGVYPPDDF------QQVKKFGLS--LMISTDA 56 H.sapiens_MAD2_MAD2L1 4 QLSREQGITL-RGSAEIVAEFFSFGINSILYQRGIYPSETF------TRVQKYGLT-- LLVTTDL 59 N.gruberi_NAEGRDRAFT_44313 5 AQ ATKNT IT L-KG ST E IVA EFFGY S IN S ILYQ RG IYPPETF------TKVSKYGLP--MMVTTDE 60 E.gracilis_Gene.95925::EG_transcript_22781 6 TQ STKNV IT L-RG STQ IVT EFFGYA IN S ILYQ RG IYPAESF------APFQHYGLP--MMVTADP 61 E.gymnastica_Egym_Gene.3431::CAMNT_0000692281 16 AQ GTKNV IN L-RG STQ IVT EFFGYA IN S ILYQ RG IY P A ET F------T T FQ N Y G LQ - -MMVTTDP 71 D.discoideum_DDB_G0273201 4 AVASKTN ISL-KGSTEIVTEFFSYSINTILFQRGLYPP ESF------TRVAKYG LP--ILVTNDQ 59 B.natans_Bigna1|92439|estExt_fgenesh1_pm.C_230012 5 TEAKSNTITL-KGSAQIVSEFFGYSINSILYQRGIYDPELF------DATKKYKLR--MMVTKDK 60 Mad2 R.humris_DN15003_c2_g6 4 AQ A EKQ V IT L-RG SV STCV EFFGYAVN SLLYQ RGVYPAECF------TTVSKYKLQ--M LVTTDQ 59 S.specki_Gene.117927::Locus_110322 3 AQ ADRTV IT L-RG SV STVV EFFGYG INT ILYQ RGVYPAETF------TTVNKYKLQ--M LVTTDK 58 Prokinetoplastina_PhM_4_TR2513|c0_g1_i1|m.55700 24 VQQ AGGQ IT L-RG SVTVVT EFFAYAVN S ILYQ RGVYPDNSF------QKVTRYGMQ--LLMTQDE 79 Prokinetoplastina_PhF_6_TR1465|c0_g1_i2|m.2639 15 QQQAGASITL-RGSVTVVTEFFAYA INSILYQRGVYPAESF------QKVQRYNMP--LLVTQDN 70 O.tauri_jgi|Ostta4|6878|gw1.14.00.313.1 1 ------A IAL-RGSTDHVVEFFGYAVNSVLYQRGVYPPESF------ERKSKYG IG--VMVTSEP 50 A.thaliana_AT3G25980.1 5 TAAAKD IITL-HGSAA IVSEFFCYAANSILYNRAVYPEESF------VKVKKYGLP--M LL IED E 60 S.pombe_MAD2 3 SVP IRTN FSL-KG SSK LV SEFFEYAVN S ILFQ RG IYPAEDF------KVVRKYGLN--MLVSVDE 58 G.intestinalis_GL50803_00100955 2 ATQTKNA ITL-RGSTA IVTEYFAYSINSILYLRG IYGQDSF------M SQQKYGLP-- LM V A K D P 57 S.cerevisiae_MAD2 1 ---M SQSISL-KGSTRTVTEFFEYSINSILYQRGVYPAEDF------VTVKKYDLT--LLKTHDD 53 H.phaeocysticola_DN31151_c0_g1 26 QSEELRMAEI-RASASSLIEFFGYAVNGILRHRRVYPPDFF------TNAQRYRLS--LPVATDR 81 C.merolae_CMS347C 4 AGSEQAP ISL-RGSTDTV IEFLRCA IYSILYQRG IYPPEM F------RRASKFGTS-- VLVAQEA 59 H.sapiens_REV7_MAD2L2 2 TTLTRQDLNFGQVVADVLCEFLEVAVHLILYVREVYPVG IF------QKRKKYNVP--VQM SCHP 58 Rev7 S.cerevisiae_REV7 3 ------RWVEKW LRVYLKCY INLILFYRNVYPPQ SF------DY---TTYQ SFNLPQ FVP INRHP 52 H.sapiens_HORMAD2 18 ETV FP SQ ITN EH ESLKM VKK LFAT S ISC ITY LRG LFPESSY------GERHLDDLS--LKILRED 74 Hop1 T.brucei_HOP1 1 MTSTATEVTQ-TQSLAAVRNFIRVAVSCVTYLRGLC SD ESY------Q PRQ FLG LQ --LKQLVRS 56 S.cerevisiae_HOP1 10 KTETKTEITT-EQSQKLLQTM LTM SFGCLAFLRGLFPDD IFVDQRFVPEKVEKNYNKQNTSQNNSIK--IKTLIRG 82

Tbrucei_TbMAD2 57 ELNSY---LAEVLQQ-V IKWVSQDKLRKLVLVLVDA-ETKGDVVERWVFD IATQGLEAV------110 T.cruzi_TcCLB.510091.40 57 DLNAY---LAELLQQ-VAMWVTADKVRKLVMIVADG-AESNVVVERWAFD ILAEETKGM------110 T.vivax_TvY486_0301060 57 ELNSY---LSEVLGQ-VAAW INTDKMRKVVV IIAKS--DDNEVIERWVFDITTEENVGC------109 L.mexicana_LmxM.25.1670 57 DLNAY---LAELLQQ-ISSWIAHGTCRRLVILITDV--Q SVRTM ERW EINIETEPAASS------109 H.sapiens_MAD2_MAD2L1 60 ELIKY---LNNVVEQ-LKDW LYKCSVQKLVVV ISNI--ESGEVLERWQFDIECDKTAKD------112 N.gruberi_NAEGRDRAFT_44313 61 QLRKY---LSQVLNQ-LSNWLLQKQVQKLVLVITSV--N SSEV LERWQ FD LEVDDN IT E------113 E.gracilis_Gene.95925::EG_transcript_22781 62 ALKKY---LGSVLTQ-LSEWLMNQVVQKLVLVVSSV--ATN EV LERW V FN IEV ESGAGA------114 E.gymnastica_Egym_Gene.3431::CAMNT_0000692281 72 GLRKY---LGNVLNQ-LSDWLLNSLVQQLVLVVTSV--ATN EV LERW V FN IEV EADAM A------124 D.discoideum_DDB_G0273201 60 SLKDY---LDNVLKQ-LSEWLLSGDVQKLVLV ITDI--VTKEVLERWVFDVTTDIPKEG------112 B.natans_Bigna1|92439|estExt_fgenesh1_pm.C_230012 61 GLSEY---LKNVLEQ-LTAWLEKGMVQKLVLVIASV--DTG ET LERW V FDVDTNKKC LK------113 Mad2 R.humris_DN15003_c2_g6 60 GLKAY---LGNVLQQ-LSEWLMRRMVQKLVLVISD---DSETVLERWQ FD IESDAVGGG------111 S.specki_Gene.117927::Locus_110322 59 GLKSY---LGNVLQQ-LSEWLMKGLVQKLVLVISG---DNDVVLERWQ FD IDTDD ITAG------110 Prokinetoplastina_PhM_4_TR2513|c0_g1_i1|m.55700 80 ALRNY---LGQVLSQ-ITDWLLQGCVHKLVVVIADQ --A SGATM ERW V FD LEPQ TAAM D------132 Prokinetoplastina_PhF_6_TR1465|c0_g1_i2|m.2639 71 ALKTY---LGHVLSQ-LSEWLLMGAVQKLVILISDQ --ATG ST IERW V FDVDADTTAAP------123 O.tauri_jgi|Ostta4|6878|gw1.14.00.313.1 51 KLREY---LVNVLER-VDQWLMESDLRKLVLVLADA--RTGATVERWAFDVQTDGSKTR----RDGENA------109 A.thaliana_AT3G25980.1 61 SVKSF---M SNLTSQ- ISEW LEAGKLQRVVLV IM SK--ATG EV LERW N FR IETDN EVVD------113 S.pombe_MAD2 59 EVKTY--- IRK IVSQ-LHKWM FAKK IQKLILV IT SK--C SG ED LERWQ FNV EM VDTADQ ------111 G.intestinalis_GL50803_00100955 58 ALSKY---LGEVLQR-VAEWLMSGKVQKLVMVLLPI--GGSEPIEKWEFNVENEGVQAG------110 S.cerevisiae_MAD2 54 ELKDY--- IRKILLQ-VHRW LLGGKCNQ LVLC IVDK--DEGEVVERW SFNVQH ISGNSN------106 H.phaeocysticola_DN31151_c0_g1 82 ALKSY---LGNSLQQ-LSDWLVRGVVHRVAVVLQG---GDGRVVERWQ FEVAELPQG------131 C.merolae_CMS347C 60 QLDSY---IERILQRHLRLWVLRGSVHRVVLAFAAT-ADPGR ILERWHFDLHM EQRATE------114 H.sapiens_REV7_MAD2L2 59 ELNQY--- IQDTLHC-VKPLLEKNDVEKVVVV IL DK---EHRPVEKFVFEITQPPLLSI------110 Rev7 S.cerevisiae_REV7 53 ALIDY---IEELILD-VLSKLT--HVYRFSICIINK--KNDLC IEKYVLDFSELQHVDK------103 H.sapiens_HORMAD2 75 KKCPGSLH IIRW IQG-CFDALEKRYLRMAVLTLY TDPMGSEKVTEMYQ FKFKYTKEGAT----MDFDSHSSSTSFE 145 Hop1 T.brucei_HOP1 57 SGDAGA-- ISRWM EEGA FDA LNRGY LK ELSLCVH EP--NCT ELLERY SFM V SY SPGGQ RAA LSLTG ESM KENLSCS 128 S.cerevisiae_HOP1 83 KSAQAD-LLLDWLEKGVFKSIRLKCLKALSLGIFLE--DPTDLLENY IFSFDYDEENNV------N IN V N L S- - 145

Tbrucei_TbMAD2 111 ------E-GV----NSKSDDTLRKEIQAVL RQ VT SSV SY LP LLHRP--CY FDM LVYADP------156 T.cruzi_TcCLB.510091.40 111 ------IA----TTRTDAELQREIQAVM RQ ITA SVAY LP LLP EG--CV FD LLVYT EM ------155 T.vivax_TvY486_0301060 110 ------G------PAVKEEEVRGEIQALL RQ IT SSV SC LP L ITQ P--CH FDT LVYTD L------153 L.mexicana_LmxM.25.1670 110 ------SRLHS--GARKSEEDVRM EIQAVM RQ ITACV SFLPV ITQ P--CA FD LLVYT SA------158 H.sapiens_MAD2_MAD2L1 113 ------DSAP----REKSQKA IQDEIRSV IRQ ITATVTFLPLLEVS--CSFDLLIYTDK------159 N.gruberi_NAEGRDRAFT_44313 114 ------NEGH----NTKDLKQ ITSEIQAV IRQ ITASVTFLPLLNEP--CTFDLLVYTSK------160 E.gracilis_Gene.95925::EG_transcript_22781 115 ------D-KA----VEKPERQ IQGEIQAI IRQ IT A SV T F LP L LQ E S- - C T FD L LV Y A H K ------160 E.gymnastica_Egym_Gene.3431::CAMNT_0000692281 125 ------T-GS----AEKSEKEIQGEIQAI IRQ ITASVTFLPM LAEA--CTFDLLVYAKK------170 D.discoideum_DDB_G0273201 113 ------E-AP----RQKPEKEIMNEIQAI IRQ ITASVTFLPLLPNA--CTFDLLVYTSK------158 B.natans_Bigna1|92439|estExt_fgenesh1_pm.C_230012 114 ------DGST----QEKSEKDIMREIQAI IRQ IT S SV T F LP L L E EP - - C T FD L L IY A P N ------160 Mad2 R.humris_DN15003_c2_g6 112 ------AA----AAKNEGAVQREIQA IV KQ ITATQ T FLP LLPD S--C SFD LLVYTHA------156 S.specki_Gene.117927::Locus_110322 111 ------G-RG----GAKSEDATQREIQA IV KQ ITA SQ T FLP LLP EH--CT FD LLVYTHT------156 Prokinetoplastina_PhM_4_TR2513|c0_g1_i1|m.55700 133 ------ASSS----VPYNEKEVQAQ IASIM RQ ISA SV SFLP L IDKP--CT FD LLVYAR S------179 Prokinetoplastina_PhF_6_TR1465|c0_g1_i2|m.2639 124 ------SSSAK--PTPYQ EKEVQTQ IASIM RQ ISA SV SFLP L ISAP--VM FD LLVYAKG------172 O.tauri_jgi|Ostta4|6878|gw1.14.00.313.1 110 -GDANG---EETMK----TEKTEKEIMQEIQAI IRQ ITASVSFLPLLDRE--CAFELLAYTDK------162 A.thaliana_AT3G25980.1 114 ------K G V S - - - - R E K S D K E IM R E IQ A IM RQ VA SSVTY LPC LD ET--CV FDV LAYTDT------160 S.pombe_MAD2 112 ------FQN IG----NKEDELRVQKEIQAL IRQ ITATVTFLPQ LEEQ--CTFNVLVYADK------159 G.intestinalis_GL50803_00100955 111 ------AT----SSKPEADVQREIQA IL RQ INASVSFLPV ISDP--VTFD IM IYTDA------155 S.cerevisiae_MAD2 107 ------G-QD----DVVDLNTTQ SQ IRALIRQ ITSSVTFLPELTKEGGYTFTVLAYTDA------154 H.phaeocysticola_DN31151_c0_g1 132 ------HM FAESSFLNEVQAVMKQ ITAA ET FLPP LEGD--CT FD LLVYTQ L------174 C.merolae_CMS347C 115 ------D-SS----VPRDESVVMREIQA I IRQ ITASVTFLPLLDEP--CSFDLLVYTDP------160 H.sapiens_REV7_MAD2L2 111 ------S------SDSLLSHVEQLLRAFILK ISVCDAV LDHNPPG--CT FTV LVHTR E------154 Rev7 S.cerevisiae_REV7 104 ------D------DQ IITETEVFDEFRSSLNSLIMHLEKLPKVNDD-T ITFEAV INA IELELGH KLDRNRRV 162 H.sapiens_HORMAD2 146 ------SGTNNED IKKASVLLIRKLY ILMQDLEPLPNNVVLTMKLHYYNAVT------P 192 Hop1 T.brucei_HOP1 129 FGTVGGGGGGDKM GKV LRKRRCRQ EVQQ A LA S IITKLMEVVEGLPPLLCERVLTMQ LKYYEDVT------P 193 S.cerevisiae_HOP1 146 -GNKKGSKNADPEN----ET ISLLDSRRMVQQ LMRRFIIITQ SLEPLPQKKFLTMRLMFNDNVD------204

Tbrucei_TbMAD2 157 DTELPT------GSW ELSD--PR-LIM----- GSSEVTLRSFSTS------FHSVSASVAYRDGS---- 201 T.cruzi_TcCLB.510091.40 156 DAELPS------NTW ELSD--PR-LIP-----CSSEVKLRSFTTN------FHRVNASVTYREDAA--- 201 T.vivax_TvY486_0301060 154 DTEMPA------GSWEASN--PR-LIL-----DSSEVRLRSFSTS------FHGVAASVAYKNQRV--- 199 L.mexicana_LmxM.25.1670 159 DAQVPS------TAW EPSD--PQ-VLK-----RGTEVKLRSFTTS------FHHVDTSVVYRE------201 H.sapiens_MAD2_MAD2L1 160 DLVVPE------KWEESG--PQ-FIT-----NSEEVRLRSFTTT------IHKVNSMVAYKIPVND-- 205 N.gruberi_NAEGRDRAFT_44313 161 DICTPF------AWEESD--PK-YIT-----QSSEVRLRSFSTS------VHKVDTLVSYKD------202 E.gracilis_Gene.95925::EG_transcript_22781 161 DCDVPA------TW EESD--AR-FIQ----- NASEVRLRSFTTK------IHRVDAMVSYRCED---- 204 E.gymnastica_Egym_Gene.3431::CAMNT_0000692281 171 DCSIPG------TW EESD--AK-Y IT-----KSNEVRLRSFTTK------IHKVDAMVSYKCED---- 214 D.discoideum_DDB_G0273201 159 DLAVPQ------KWEESD--PK-FIT-----NSQQVKLRSFTTT------IHKVESMVAYKISND--- 203 B.natans_Bigna1|92439|estExt_fgenesh1_pm.C_230012 161 ECKVP E------KW EESD--PK-Y IA-----KSNEVRLRSFTTK------IHKVEASVAYKLS----- 203 Mad2 R.humris_DN15003_c2_g6 157 DADVPV------SWELSD--PK-LVV-----DATNVKLRSFSTH------VHKVDTSVTYRH------198 S.specki_Gene.117927::Locus_110322 157 DAEVPP------AW EVSD--PK-LVV-----NATNVKLRSFSTH------VHK------189 Prokinetoplastina_PhM_4_TR2513|c0_g1_i1|m.55700 180 DTSVPM------TW ETSD--PK-LIV-----DSTQVKLRSFTTT------VHKVDTSVCFRE------221 Prokinetoplastina_PhF_6_TR1465|c0_g1_i2|m.2639 173 DTVVPQ------TW EMSD--PK-LIT-----NSTQVKLRGFTTT------IHKVDTSVCFRE------214 O.tauri_jgi|Ostta4|6878|gw1.14.00.313.1 163 ESATPD------ECEESD--PK-YVR-----DGVEMRLRSFSTK------IHKIEAGVSYK------203 A.thaliana_AT3G25980.1 161 DVAVPF------TW IESD--PK-LIA-----NPQMVKLHGFDTK------IHKVDTLVSYKNDEWDEE 208 S.pombe_MAD2 160 DSEVPT------DWVDSD--PR-ILR-----DAEQVQ LRSFSTS------MHK IDCQVAYRVNP---- 203 G.intestinalis_GL50803_00100955 156 AVPTPA------EWEECP--GRDHVH-----NAVEVKFRDFSTK------IHKVDTAV IYKSGEEAL- 203 S.cerevisiae_MAD2 155 DAKVPL------EWADSN--SK-EIP-----DGEVVQ FKTFSTN------DHKVGAQVSYKY------196 H.phaeocysticola_DN31151_c0_g1 175 GTTHPA------TW EMST--QR-LVA-----GSSDVHLRAFSTG------LHQVSTSVSYLAD----- 217 C.merolae_CMS347C 161 ALEADA------NEWEESD--AK-LITA---PRCEQVKLRSFTTN------VHRVETGVAYACKELTDT 211 H.sapiens_REV7_MAD2L2 155 AATRNM EK IQV IKDFPW ILAD--EQ-DVHM---H DPRLIPLKTMTSD------ILKMQ LYVEERAHKGS-- 211 Rev7 S.cerevisiae_REV7 163 DSLEEK--AEIERDSNWVKCQ--EDENLPDNNGFQPPKIKLTSLVGSDVGPLIIHQFSEKLISGDDKILN- 228 H.sapiens_HORMAD2 193 HDYQPL------GFKEGV--NSHFLL--FDKEP INVQ VG FV STG------FH SM KVKVM T EATKV ID L 244 Hop1 T.brucei_HOP1 194 PSYEPP------CFAPAS--PS-TVAAHYREEKN STD IAT LDTG------HHT LS IN IRHK FFEQ LR- 245 S.cerevisiae_HOP1 205 EDYQ P E------LFKDAT FDKRAT LKVPTN LDNDA IDVGTLNTK------HHKVALSVLSAATSSMEK 260

Figure S1. Sequence analysisFigure of TbMad2 S1. Sequence analysis of TbMad2 Multiple sequence alignment of Mad2 from various eukaryotes as well as Rev7 and Hop1 from some species, showing that TbMad2 has higher sequence similarity to Mad2 than to other HORMA domaining containing proteins.

21 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424754; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Putative Mad2-interacting motif T.brucei_Tb927.6.1220 4 ASQ---EFALLQHLKVSIPNDPVP------PQPKAQ AVRNTHRVM SPCRRPP------VSQK 50 T.cruzi_TcCLB.507603.60 1 MSQH--EFSLLSHLGVSVPEVPSQ------TVQTNNRREGNYRVLAPCMRPP------VKGA 48 T.rangeli_TRSC58_05191 1 MSQP--EFLLLSRLGVSIPDASPQ------ALQTTRSCEGRFRVLAPCMRPP------AKGA 48 T.grayi_DQ04_01761100 1 MTQ---EFSLLHHLGVSVPDRASP------TPQVTGSRDGNYRVLAPCKRPP------VKEG 47 T.theileri_TM35_000321090 1 MSE---EFSLLDHLAVSVPEPA------LTAAERPQQYRVLAPTRR------37 T.vivax_TvY486_0600730 61 MSQ---EFSLLQHLGVTVPESVAR------NVLATTPVRNTFRVM SPCRRPPG------AVGA 108 Kinetoplastids L.mexicana_LmxM.12.0070 45 MSG---EFSLLQHLGVTVDRCDSS------ADRAPPTHSAKAAFRVWAPTQHPP------SKC 92 E.monterogeii_EMOLV88_120005700 1 MST---EFSLLQHLGVNVEKCDFS------RSVAPTTSSEKETLRVWAP IQHPP------TKS 48 L.seymouri_Lsey_0135_0090 1 MSA---EFSLLQHLGVSVECGLAPAEQ------RSGAAKTPPRDTLRVWAP IRHPP------SNKN 51 C.fasciculata_CFAC1_010006300 1 MSA---EFSLLQHLGVSVDKCEAPARATA------SSSR------AAAAAAAAPAKNAFRVWAPAQHPP------INKA 58 B.ayalai_Baya_067_0260 1 MS----EFSLLQHLKVVIPECAPA------LSGAKPPPAASHYRVWAPVNRPP------RKG 46 P.confusum_PCON_0057390 1 MAT---EFSLLTHLGVQVPEATAVTTA------PPAQ------GLSAVVGPHHAGHFRVLAPSKRPP------QPRE 56 Prokinetoplastina_PhM_4_TR15442 1 MS----EFNALAFLGVSSSASVTV------AAPPSSSCAAGVRVLAPK IKAAE------YERQ FRSA 51 E.gracilis_light_m.40759 1 MA------DLLSYLGVSIPAEPTR------KAAHAQPPPPPAYFRPRHAAPPPP------Q SRG 46 Euglenids E.gymnastica_Egym_Gene.29462 18 ISCT--M CD LLGY LGV S IP SEPAGV SSHAG SKD FSFYKPSCNTN IVSSSGARTSRDRLGSQCK IEIPNLRNADQQDQATTPKCPPP------101 Diplonemids R.humris_DN13470_c0_g1 57 MSSTDGGYDMLGQLGVVVPSTRVVPPA------PSADARAARREPRVMKPSARAPQPAVSTASASASASHRSHHHHHRRT 130 H.phaeocysticola_DN13286_c0_g2 12 FTS------AVAPPPAPAPPPS------GDPPPLAAAAG EG SVT LPADPPPQ PHPQ P LPHRTDNRHDQ PYLSPRRQ 75

T.brucei_Tb927.6.1220 51 VAR IATAVNT---KKELQAPRA------PM A EFSG EV------M TVVGKN------NRPNVEYLIYDAKIPSRNA INMQ LAEVKKQRAMGEKVR 123 T.cruzi_TcCLB.507603.60 49 I---AAQV----- ISPRRVPRV------P LADYTGEV------VVVKGKG------GRPNTEYLIYNARVPC IRAVNLQLMKERRRQREEEAVS 116 T.rangeli_TRSC58_05191 49 M---TAQM-----TSPRHLPTA------PLADYAGEV------ITVRGKG------GHPDTEYLIYNAKSPSIRATNLQLMEEQRRKREEYAVS 116 T.grayi_DQ04_01761100 48 T---AAER-----STKRR IPTA------PLADFAGEV------VEVRGRK------NQADTDY IIYNARVPSIGAANMQ LITEGQKRRTEESLY 115 T.theileri_TM35_000321090 38 ----AAEG-----PQRRQ LPKA------PFADFTGEV------LTVKGKN------EAADTEY IIYNSR IPSIQAVNMQ LIEEKR KRHVEGN IC 104 T.vivax_TvY486_0600730 109 V---SAKMH----ARGRSAPEA------PLADFAGEV------MTVQAGK------GGNDTEYLIYNARVPSVEAVNMKLAEERKKEHRDKRNE 177 Kinetoplastids L.mexicana_LmxM.12.0070 93 G---TAEETCRVLEKKRKGAHS------SYADPTGEV------LLRRK------NGVEVEYVVYNSK IPSVHGVNKRLAETK-AEREREENS 162 E.monterogeii_EMOLV88_120005700 49 G---SSQEARLAREKNRKGVHH------HYADPTGEV------LLSHK------NGAEVQCLVYKSRVPSVYGVNKQ LAEKKKAEREQVKST 119 L.seymouri_Lsey_0135_0090 52 V---SVDQVSGVRDRKRKGPQK------SYADPTG EV------LLRK E------YGAEVQYVVYNSKVPSIYG INQQ LAERKRAERARSSSA 122 C.fasciculata_CFAC1_010006300 59 I---PTERARRALQHNRKGPHK------SYADPTG EV------LVRH E------HGAEVQYVLYNSKYPSVHGVNKQ LAERKAAERTRESAA 129 B.ayalai_Baya_067_0260 47 I---SSDMRS---PQRPRPPHN------K CADPTGEV------IFGCE------NGQPVEYTVFNAK IPSVYGTNLLLAERKRQ ETTAR LEG 114 P.confusum_PCON_0057390 57 A---PTDRN----PSRHLPPPP------QAATIAGEV------LIPKGGG------AEYV IYDSLVPSTQGVNLQLMEQKRQQKQRQRGR 121 Prokinetoplastina_PhM_4_TR15442 52 S---ARGTATGRPTAGSKMPSSVGASTPRP IQNN SNA LKAA EP------IPRA SA SPQ PG IAVAPTNERVEIYEGPCPSNLA INQQAAREREEARARQ R ER 143 E.gracilis_light_m.40759 47 P------TTPARPPAT------PSASPSQEG------TPLRSTFQRRV------RMADVEY IVLDSPVPCNMAVNARLAEERRER ERQQ RQ A 114 Euglenids E.gymnastica_Egym_Gene.29462 102 ----AEPR-----DQQDHLPKE------RSKDMAAESKSGNSFLQVALDHKVETIRRRTK------RGGYVDYD IYESKVPSNLAQNMALAQQKKER EKVVR EQ 184 Diplonemids R.humris_DN13470_c0_g1 131 S--- SSGTR SLSPARV SDVP SACR SG LHR---RHTVAEEEGET------VLRM EGG------RGGDDEYLVYDATVPCNLAVNAGLSGAHAERARAQ AQ R 212 H.phaeocysticola_DN13286_c0_g2 76 AW -GAGHTTQ PVPTRPD LVPHTT SD------RVFKEEGRT------VLRRKGA------RGEVQ EFEVWDAGVPSNAVYNELAARSRLDSANEERMR 153

T.brucei_Tb927.6.1220 124 HR--CHR------HEVD-AWGERDDAKCKQRLLKSLQE--VNMEQAHRTRAEREAQQRRRMETEANGIESYTVACNA LDP EKT IEG-KKA LRDQ LQQ AA FESLDNHK--K 219 T.cruzi_TcCLB.507603.60 117 RI--SPD------TFFI-QTSERE-DLARRRLLEH LQ N-- INR EQ AQ RKV EEK ER ERQ RR LEV EVA ELKAVK E-AAVN EAAA LL IK-KQ AARQ AMQ R ELLDAM ERKR--Q 210 T.rangeli_TRSC58_05191 117 RM -- SPD------K LL I-D SQ ESN-D LARRRV LEH LQ R-- INR EEAQ RKV EERAR ERQ RR LER ELAD LRAV EE-A SAK EAAD LLVK-KQ A ERH FMQQ AV LEATERKR--Q 210 T.grayi_DQ04_01761100 116 QL--SPN------VEYL-QPHQRS-DAAQRRLLTM LQ N--ANR EQ AQ RK LEEKQ R ERQ RR LER ETA ELKR IEA-STVKDDFA IAEK-KAAEREYLKQAALEAV ERKK--Q 209 T.theileri_TM35_000321090 105 DK--SPN------IAFF-KSREREYALRRRRLLENLQR-- INM EQAQRKM EERQREREKRLGRP IMK LKVNSE-EPNREVVTT ITRNNKSESEA IQ SNVLV ETDGKKD LD 202 T.vivax_TvY486_0600730 178 EL--R ID------DAM F-GNRTSD-EVKRRQ LLQ ILQQ --ANM EQ ARRKR EEK ERDRCKR IEH ER SELRRLAENESAAETARAAER-RKKQLEELRQLAEQSRENKR- LR 273 Kinetoplastids L.mexicana_LmxM.12.0070 163 PY--VMC------GLAL-GKGEAGQ EEARRLLLQELRR--CNQEQARMKKEEGLRERARRIEHERAVIQYNAG-EAAREEGNATQK-KKMDGEAVSEAAVKA LA LR E-- E 257 E.monterogeii_EMOLV88_120005700 120 PG--FGN------GLLI-GNDEGRREEARRELLRELHR--CNQERARLKKEEELRERVRRMEQERLVLQYNVG-RATPREDEVLQ E------GAVRDAVAEGPSRRE--A 209 L.seymouri_Lsey_0135_0090 123 TALETYE------LEF-GQGEALREEARRH ILRGLQL--CNQ EQASRKKEEERRDRERR IALEKAHLRY LDA- EAART EEEAR LH-RAHQ EEV LRKAAAQ AAAAKK--H 218 C.fasciculata_CFAC1_010006300 130 AANAVHN------GLVF-GEGEALQEEARQQ LLRELYE--CNRDQANRRKEEERRDRERRIAQEREQLRYRDA- EAAR SEQ EAHM H-RVHQ EEAM RRAAA EAVAAKK--R 226 B.ayalai_Baya_067_0260 115 EQ R C T G E E- - - - - V ST SY - - G N EV IR ET A R R R L LQ QLQQ--CNRDQAERRREELQREAVRRLQKEREHLV SV EA-D EKRR EQ N EM EA-KRDVQ A SRRKAAA EAM LAKH--M 212 P.confusum_PCON_0057390 122 SE--AHHQDPNGSASFFLGGPGDLSVAARQRQVLAELQQ--HNMEQARRRKEEQQRAAAARCAQERAWLQ EAQQ-ESAHEAEQ-----RRLQRRAASTEARD NASRLR--S 220 Prokinetoplastina_PhM_4_TR15442 144 DR------EATMQQ LAL------AQRVAAEERQKKLQEREATRTSW------DAYMHNVEESRH-REARAQRHQKEAEQALIAAS--- 209 E.gracilis_light_m.40759 115 EVA------AAAEQD-ATQRQW LQQ TLQQTLSAREAAQQQLQDFQRSHAEAAEH------AKAEEMKQQQQVRD IQ D R I L - - - 181 Euglenids E.gymnastica_Egym_Gene.29462 185 DR--AQA------QSVSE--ANAQWAERVWQEYQTQKTLRRQELR------DFQVLHEKYVQV-ARAREERQKQKEREDM GRQ A--Q 252 Diplonemids R.humris_DN13470_c0_g1 213 DA--ALV------DKARAADAAYAVE------KKRAAAEGRRARAAEVRSARAVQEAHAA-RASSAGAAAL---RREREQATEAYARAVEDKVQ--- 288 H.phaeocysticola_DN13286_c0_g2 154 EQAFLEAQ------AAAEVAAEQGRQHAL------LAQQ EAARVETVRWNADRKAALER------QREREREEAAFFRDAQ DRRA--- 220

T.brucei_Tb927.6.1220 220 GEPVEGAAWDLHYEPFPWNN------DTRRKR--- IQRDRCVALMDANRELAEKKRDERQRQRAM EAA WNYGGGKGVSFGGKR------YAHDVGRNPSLQAARGSDDGAV 315 T.cruzi_TcCLB.507603.60 211 EAADVNNHVA LECAA FPW DN------APPN EA---KRREICLSLLDANRKLAEDKKM ERQARK IEERAREAAALEVVRAEVER---EDRRALEKKRYLCEERQRSGMGTFV 309 T.rangeli_TRSC58_05191 211 EAADVNGG LAAGCAA FPW D E------V SQ NGA---LQREKCLFLLDANRKLAELKKIEQQTRKIEEKVREEAVLAEVRTQAEK---EKRKALEKKRRLAEERRSASVGAQV 309 T.grayi_DQ04_01761100 210 RAAEANSRGEVTCTAFPWDN------PPQDGS---RMRTQCLALVDANRELAEQAKQRKELTQMKERAKENAAVARTQALIGE---ERIRELRKKNELREMRRCKSAEPYE 308 T.theileri_TM35_000321090 203 TESEFKGRNELVCSAFPWDN------ESQREN---NRRAESLA IM ETNRKLAEDAKKEQ EMKKMKEISM ELAALEENRAK IEE---ENARLQ EKKRALQR IQ RSCSFNTHK 301 T.vivax_TvY486_0600730 274 PTSSRNSQDGKECFAFPW EA------PASENK---GARARC IAIREENRRLAEQRRNQHECLG---SARSPAAEKFIATDAGA------VDGGGDTSPQAAHVADNMGV 364 Kinetoplastids L.mexicana_LmxM.12.0070 258 RRADAPVESRVV--ASGWDG------KDEDDRRRLAAA ERTRQ LA EEN FRAA EQ RRA ERM AQ EQ GAR ERAQVERM ELQRQ LAH---EHEKDLERHRRNAEALRGAQ EAARE 357 E.monterogeii_EMOLV88_120005700 210 REDDAKKFARGA--SMAWNW------KDEDDRRRAAAAARTRQ LAGENLLAAEERRAEHKALKEADRKKAHAEVTKMQMQ LAE---EHTREMEKHRRDAEELRGALEAARE 309 L.seymouri_Lsey_0135_0090 219 AAETSKNVDD IC----VWAG------ADEDEQRRRASQRRTRECAEANQRMAERQRAERNASEAAERERSRVERAEVERQARE---ARTREAAKQQRAAEALRREQEAERE 316 C.fasciculata_CFAC1_010006300 227 AVAAAEDAECLFY-AGAWNG------ADEDERRRCEALRRTREFAEANHRTAERQRAENIAAAAAERERARELR IVVEQQLQE---EQKHEAERHRRAADALRSAQETERE 327 B.ayalai_Baya_067_0260 213 RLAAENERAKEVATGVILSPR-----VAVSAA---EERARC--IAEENRQISEKIKKERLAQRQRELEEARAKAAW EEEQ TRRAGMQ CKR EK ER LRAY FEELADMAEKDKA 313 P.confusum_PCON_0057390 221 RQQ DDTAV SQQ SC--Y EW ESSG LGA LSA LSAAR EEA SVRRDR ETM EANRR IAD SR SA SR SMM ERHARDAAAAAAAALQQSTTEAAEGRAASRRAKQQM LDELRGAAEASAH 329 Prokinetoplastina_PhM_4_TR15442 210 ------RSEAEAEARRLQEKKNVTAEDLRHQ LQL---EINRKAAKKAQNDEEERQ R LFNAT L 262 E.gracilis_light_m.40759 182 ------QADREAEQQQLAARRHAAQR------LAEHQRQTAALAEEQRAQ AKA EGQ A 226 Euglenids E.gymnastica_Egym_Gene.29462 253 HSQA------EARLKREQEQQQVRAFQQAQAQHAALWTEA------RKQQREAQKKQ SSTSLG----- 303 Diplonemids R.humris_DN13470_c0_g1 289 ------KVAAARAAKDS-YRGDLTS---LLERKREKDAAEARHRAKPG IDG-F 330 H.phaeocysticola_DN13286_c0_g2 221 ------ELFEDAERRREREIREGKSE-WRAELDSH I----RERQRLAQQQRDE EQ AM A R G D F 271

T.brucei_Tb927.6.1220 316 EEGAAAL----SFG------QPTEC----FFRFEGDNPSVRWNECRKLAEVNK--Q LAEQ A ER IR K G ER L ER IEG EK K FNQ Y I- - - ER IER E EKQ SRRA EK 397 T.cruzi_TcCLB.507603.60 310 KAAPVVL------ETSDLG SEW FLRTTR EDAGARR ERQ R ELM EANK--RM A EETKRRRAEEVERR IQQ EREVLREA---ERAYTQ RM ECDW A EK 392 T.rangeli_TRSC58_05191 310 KTAPAAV------GPSDRSTCWFLQSTGEDAEARRARERELMEANK--QLAKETKRRRVEDAERQLQEERRRLQES---ERAHMQEM ER ER A EK 392 T.grayi_DQ04_01761100 309 TRAAQQV------GYDGSYDYW FAR SR- SDGYARR EK ELK LR EENR--R LADEARKRQAEETARRVQQEKEMMARV---ARFDRAESHRQ LAEK 390 T.theileri_TM35_000321090 302 ENATTRV------KDSDTFWDW FPRPKTADENRTREKERK IM ETNR--KMA DEAKRRR IEEEELR IQ SERNELREA---EELYQK QKERKIIEK 384 T.vivax_TvY486_0600730 365 KEHARCA------TPRHEDSGDGW FFRTHAA SPRCAR EKM SK LM EENK--RM A EEAKNRQ IQ EKLQR IKSEQ EFLRRA---SEAYKE EVM RQQ R EK 449 Kinetoplastids L.mexicana_LmxM.12.0070 358 LRSRADAAD------AHLKAVPSG SLFDAVERRQRDEAMRAQQKRM EDMAVNV--RLAAQ KRANAQ A ERDR---- ERQ YAA EY---AKA ELESFQREVEHA 443 E.monterogeii_EMOLV88_120005700 310 SQCRGNAVD------ACRQAGPSG SLFDEVERREREAAVQARQRLADDLAFNL--RLAAQKRESIQAERER----ERRLAADR---ARAEVE SLRCDMAHM 395 L.seymouri_Lsey_0135_0090 317 RRLVTSAAG------RRASPAPVGSLFDFAEQRESQEALRAQQRLREDMA INA--RLAEARRTQ AR EER ER---- ERR EAARV---ADAN FADFQREVAQS 402 C.fasciculata_CFAC1_010006300 328 RRLTATSRR------GRVSPAPVGSLFDFAEQRESAAAQRAQQRMREDMA INA--HLAEAKRTQ AK EER ER----DR LHAAQ V---A EAN FEDFQREMAET 413 B.ayalai_Baya_067_0260 314 RAREQRERE------V----HYSAVPLIPDRE------EEAR EKAQ R LRQ LSD INR--R LAAEKSECVRLERER----ERQAVAVS------377 P.confusum_PCON_0057390 330 RKQQQRERERQTAGLVW EVNTADHTAGVSVLPSGS------ERDHVQ R LK LRT LQ A ENA--R LAAERKTSRALAAQREAEADRVRLQEA----RALLDAEEAKLRAQ 424 Prokinetoplastina_PhM_4_TR15442 263 VEAQGRQ------RDLET------ERHQRAALRSSWELQ-----QRVRAQRREEEEMH----ELEVNAV------AQRQAAEKAAEED 323 E.gracilis_light_m.40759 227 TVLTSLPLQ------AWD------VPM ET------LHAHRQQ LQQSLVQQ-----M EERTQRAAERKAA----DAAADART-VEQAARLEAAYAAQ EAG 297 Euglenids E.gymnastica_Egym_Gene.29462 304 ------MPPEA------QVPETV IAANRQRLAAYWKDQ----- IELRAQ EQ R EQQ EE----RRRA EQ EVA LAYGA ELSA LSQ R ER EV 369 Diplonemids R.humris_DN13470_c0_g1 331 EIGTHAA------KPREE------VVR EGAA LRRVW DQQ LEAKH EAERARRAAERRAQ----QREPQALA------HVPAVREGEKEE 396 H.phaeocysticola_DN13286_c0_g2 272 EIGARRA------MPPEA------TRAQQRAMREAWDVQ-----RAGKAARSAAEQEQ----EARFSAL------Q LDAVQREVLEE 331

T.brucei_Tb927.6.1220 398 L-----KRQ KAM SM VTRDA SKKK EG LKKV SSAN ECH--DKF---LL--YSAHEREEKNRLKREQEFYDNLEKHIASSREAQRAAKDRALEEEKRLLQETISETKRYTEEEN 496 T.cruzi_TcCLB.507603.60 393 R-----RQQDELEKEARKTAVERHEKKR I----STE--DDLCLSLF--QDNCRKAEKEKQRREQLYCEELKREVDMARESRREEKEREAMQ EKKYLEMQEIEAKRALERER 490 T.rangeli_TRSC58_05191 393 R-----KQQAAVNQAALKAAAERHEKRQ E----SHR--DELDLG IF--HDDSSKVENERRRREQLYRDELKRG IEDAREARRGAKEREAVEEKRLLSLCETEARRTLERNR 490 T.grayi_DQ04_01761100 391 R-----RQQ ELVSKAALEA ISRRRAGAQAHRHKSLR-- ED L--- FF--CCDD SK IAK EKRW K ERCYYDDLKKFITTAQETRVGEKERDVAREKRLVQDANAAARRDAKRYR 489 T.theileri_TM35_000321090 385 R-----KKQ EQFSKDVLNSILEREVRRHALMDETRR--EDV---FF--RD IGSKYTKGRQ EKEQHYYDCLKKDMESAREARLEARERELEYEKRLLQESEKKAQ LDR EKKQ 483 T.vivax_TvY486_0600730 450 K-----KRYELRSQEKPGEGSVRSRARRSASVDAVQ --DG F--- FL--M FDNRKA ETGRR ERQ KRYYNALREEIASAREARIAERKNAIEQDRIFCREAAAAEK ELA EK EL 548 Kinetoplastids L.mexicana_LmxM.12.0070 444 RQ RRQQ AR LELQ DAA EAAAKVRQ AHADAARQ R EQ SM - - EP L- - - L FW P A AQ SP G A EK A K IA E SR R FR EDLCRQAEKKRNERAQEEEAERARERALIEYDTRSAQ EAV ER ER 549 E.monterogeii_EMOLV88_120005700 396 REKRHQEMLQLQQAAEVAAKHRQEHADPAQRWEQSM - - EP L- - - L FW SP A R A SQ S E E FK K A K A R R FY EDLQRQAEKKVQDRAQEREAERAMERALVDYDIRSAQ EAAAH EC 501 L.seymouri_Lsey_0135_0090 403 RLRRMQERKELRDAAEVAAAVASEREKQNALVDRSQPDDSL---LFWARDDSLLAGEAKAEAQRRYRD EVRR EA ERRHA ERAR EER EERAW ERA LV ECDN EAAKEAAARER 510 C.fasciculata_CFAC1_010006300 414 RLLRHEERAELQRAAEAATAQARETRQRAARVDESRPDDA L--- LFW GR ED SVRAA ETKA EAQ RR FYADVRRQADRRAAERAAEERAEREREKALIEFDNEVARDVAARER 521 B.ayalai_Baya_067_0260 378 ------HGSCASVHVDRQ ECQKKFYEELRREAETRREQRLAEAKRERLVERNALEEAGKAEERATAREK 440 P.confusum_PCON_0057390 425 R---RQQGEELQRAAEEA IASRREQRRREREHDAAP--SSA--GLL----SDRDAGGQ SVAAKRAYYEELLEQ IEAQQQRAARDRQATM ERQKQDAAELTAVAAAMKASEQ 524 Prokinetoplastina_PhM_4_TR15442 324 R------RMRAEAQRRANLRRDLQRTVETAHEHRHLEAQRRRDEERRAVAEELEAARRRDAQQR 381 E.gracilis_light_m.40759 298 R------RQAQRDQLYRYYEAVQHAKAERLREEHGLKQAAAEARVAAEA EA EGQQ R 347 Euglenids E.gymnastica_Egym_Gene.29462 370 K------QQRTRETLTAYALAM------KAKEERVQQRKVERQKAAAARALQDQQV 413 R.humris_DN13470_c0_g1 397 R------RARAAGLLEAQQ GQ LEEQQ AR EAARRAV ETAVDM RRVVRDHAAQAEVEAREA 449 Diplonemids H.phaeocysticola_DN13286_c0_g2 332 R------AAYSARRSAREAGRGELLAQASERRERMRREREAEQ IMDSAR IESTARV AQ EEAAKRH 390

T.brucei_Tb927.6.1220 497 R------KAKQKREAYKAALEKQ IREKSNEASHS------SPMPHRG------PEVRVLYRCPVTKDLLPPEEF-VPAAS---LRRTSTLW------565 T.cruzi_TcCLB.507603.60 491 A------KALEKREAYRKALEEQ IQ SRKTKPA CQ------APVTRHG------SVLKVLYRCPVTGDLLPPDQFGIFSVR---QRQSGWI------559 T.rangeli_TRSC58_05191 491 V------RAAERREIYRKALDEQ IRSKKTETTCE------VPATHRG------PVMKVLYRCPVTGELLLPAQFGISSTR---QRRSGWNW------560 T.grayi_DQ04_01761100 490 E------RAAQ KQ ESYRM A LQ GQ IESRKVNG ERR------SIAPHRG------PEMRVLFRCPVTGKLLTPDQFGVTSPYSFGSRRTL------559 T.theileri_TM35_000321090 484 K------RAKQRREFYMRALEEQ INAKETKNLLRH------IPPPHSG------PERKVLYRCPVTGELLPPEQFGIPLSQLQSFRRRFQ------555 T.vivax_TvY486_0600730 549 L------KRKQ K ENNYRTA LEEQ IR SKRKAVT SE------TPLRTRG------PN IGVLYRCPVTSKLLPPGEFGVPFRR---RGQNSLLY------618 Kinetoplastids L.mexicana_LmxM.12.0070 550 K------DTREKAEHLRRVLEAQ IAQKRKGTVGSRQACAAV------DVSHVPA------TEPM ILYRCPVTG ELLPA SAYD FGVQ R---GR------620 E.monterogeii_EMOLV88_120005700 502 K------EARKKAEDLRHALESQ IALKRAGM ERKEDTGAHL------SVSHVPA------TEAM LRY ICPVTG ELLPA SAYD FGVQ S---GR SRTRK------577 L.seymouri_Lsey_0135_0090 511 R------EACEKTAQ LRRTLEVQMAAKKTLQQQECSHSQRG------EPLRVPG------PEVKSLYRCPVTGELLPASAYDFG IHK---SR------581 C.fasciculata_CFAC1_010006300 522 K------VSHEKVAHLRRTLEEQMAVKKR-EERRSERLAQG------DVLRVPA------PDVM SLYRCPVTHELFPASAYDFGVRR---GG------591 B.ayalai_Baya_067_0260 441 D------RLEKQRDYLRRELDRQVLLKTGPRQRV------PDSGLRVSTPSAKKKLYRCPVTGQLLRPAAFGYSTRC------505 P.confusum_PCON_0057390 525 H------SRRVARLQYRHALDAQVAQQRET IATEARTP------RPGGADGLERRVGPQRRILLRCPVTHQ LLPPT EFD LPARH---RA SHHHR EQ VDSLLAR 612 Prokinetoplastina_PhM_4_TR15442 382 Q------AEHERQERLRADIARQIEEKVAVSAAR------RRHADPMVP------LVAEPAAR ILVRCPATGR LLPP EH FN LNAAT---ARNT LKRVQ ------458 E.gracilis_light_m.40759 348 R----- IVEETRRRVQRRGADLRQDVARRQDEKEERRRQ EA EV ERRVM ------EAA SVRPQ ATLLRCPVTGLVLPPSCFNVVIKD------R 423 Euglenids E.gymnastica_Egym_Gene.29462 414 QWDCEEVCALAQQQRKAHVTAMQAHVKANVALKAEAKQ RAW A EDRRRQ TAGPP------V EKKVLVRCPQTSLLLPPDCFNLVVTT---ATTSSL------499 R.humris_DN13470_c0_g1 450 A------CRSEKKDALKTVLGRQ LADRSAAATERADK EAAA ERR-----RPANHNG-- LTAQ KGRKVLLRCPATNRLLPPSAFNMVIRP---EKKVIV------D 532 Diplonemids H.phaeocysticola_DN13286_c0_g2 391 R------ERAQVTEERRRTLVEQ IIDKASRQQ EAAAR E------RD LEVQ RDRART LR SRR SLFRCPETNALLPPEHFNVRHTKPR- IRRSFHVGGL-Y---- 475

Figure S2. Sequence analysis of TbMBP65 Multiple sequence alignment of TbMBP65. Its putative homologs are found only in Euglenozoa species. A putative Mad2-binding motif and coiled coil prediction for TbMBP65 are shown.

22 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424754; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

T.brucei_TbAUK3 66 ------NHAADRSMW SLDDFV IIR-KLDEGRFGKVYLAREKQSKCAVVLKCISKDMIRFHS----LAHQLQREVELQEYAG 135 T.congolense_TcIL3000_9_260 60 ------GSPVKRTMW SFDDFIIVR-KLNEGRFGKIYLAKEKETKCAVVLKSISKEMIRFHG---- L A H Q LQ R E IQ LQ E Y A G 129 T.cruzi_TcCLB.510349.80 1 ------MW SLDDFD IGR-KLGEGRFGKIYLAREKRTKCAVAIKCLSKDMIRYHG----LAHQLRREVELQEYAG 63 T.grayi_DQ04_02681080 65 ------LESGPSRMW SLDDFEMGR-KLGEGRFGKIYLARERRTKCAVVLKCLSKDMIHYHD----LAHQLRREVELQEYAG 134 T.theileri_TM35_000021280 108 ------YETGKKRMW SLDDFD IAR-KLDEGRFGKIYLAREKSTKCAVVLKCLSKNMIRYHN----LFHQLRREIELQEYSG 177 T.vivax_TvY486_0900240 64 ------SIVQPPPMW SLEDFEVLR-KLDEGRFGKIYLAREKQTKCAVVLKCISKEMIRFYN----LAHQLQREVELQEYAG 133 AUK3 B.ayalai_Baya_064_0240 373 ---TPPATPPQRCAKADNWCLEDFDVGR-K IGEGRFGKIYLARERRTKCAVVLKCLSKEMLLYKD----LLHQVRREVELQAYAG 449 P.confusum_PCON_0051580 27 ------TDRAPPRQW SLDHFDVGR-KLGEGRFGKVYLARERRSKCAVVLKCIAKEMIVLNS---- L E Y Q IQ R E V Q LQ A Y C G 96 B.saltans_BSAL_23410 43 AGPSPSPDQAEMLQRRGSWTLADFD IGP-KLGEGQFGKIYLCREKTTKYAVVLKLIAKEAVIHND----LTHQIQREIELHMYC- 121 Prokinetoplastina_PhF_6_TR32404|c0_g1_i1 205 ------TETPPKNRW SVHDFQ IGK-K IGEGRFGKIYVARVQLTGHVVALKVMSKDFIVAQN---- L I LQ L K R E IN IQ Q H C - 273 Prokinetoplastina_PhM_4_TR2362|c0_g1_i3 79 ------DKSHTKPRW SLSDFEVGR-K IGEGRFGK IYLARVRLTRHT IALKVLSKDFIIQQD--- -LLLQLKREINVQQHC- 147 L.seymouri_Lsey_0037_0280 448 ------SPSRNAASWSLNDFD IGR-R IGCGATGRTYLAREKQ SKVVVALKCMQKATLASLHGDTDVMRYVTRAARLHM SAG 521 L.mexicana_LmxM.26.2440 280 ------SPATAARRW SLSGFD IGR-R IGHGHSGKTFLAREKRSKVVVALKVFNDDYAQRHDG---GASMFERAMRLQAAAG 350 E.monterogeii_EMOLV88_260028700 532 ------SPM SMSTSFSLQSFDVGR-R IGQGYTSKTFLAREKRSQAVVALRVFDKSIVQRRDG---GAHLLKQGTRLQMAAG 602 T.brucei_TbAUK1 16 ------LPPTPKSKWKLSDFELLH-KLGGGNYGDVHLASVKDCNFVCALKRLSIKKLADFD----IATQLRREIEIAFNT- 84 AUK1 B.saltans_BsAUK1_BSAL_09935 23 ------KPPAPRASWN INDFELLH-KLGGGNYGDVYLASVKNSNFIVAIKKLEIEKLVNFD----VVNQ LRREVEIAFNT- 91 T.brucei_TbAUK2 64 CVRQP---KPKVV IQQ SDLRDEQ FQRLS-VLGEG SY SVVVAARH LP SRQ VVA LK ELSRQ R LRDAK----LETQLQWEINLHRTL- 139 AUK2 B.saltans_BsAUK2_BSAL_46390 16 ------N IPVKVKQ EQLHEGSFRRNRDVLGRGSY STV FSATHV SSG SLVA LK E ISLQW ICNPA----VKDQ LKHEINVHRRL- 87

T.brucei_TbAUK3 136 RYHKNVLRLFAYFWDDVR IVLVLEYADSGTLQDLLDCYR EK EQ R- SS-- LPDDKVRT ILLQ LLSA LA FLH ERD IIHRDVKPDN IL 217 T.congolense_TcIL3000_9_260 130 RYNRN ILRLFAYFWDEERVVLVLEYANGGSLQDLLDYHRQ CQ P E-PY-- LP ENKVR IILQQ LLSA LA FLH ER E IIHRD LKPDN IL 211 T.cruzi_TcCLB.510349.80 64 RCHRH ILRLFAYFWDDVRVFLVLEYADGGNLQTLLD----TRTQ -HR--V SEEEARR ILRP LLSA LA FLH ERDV IHRDVKPDN IL 141 T.grayi_DQ04_02681080 135 RYHRHVLRLFAFFWDDVRVFLVLEYADGGNLQKLLD---- SRPQ -Q R-- LSED ETRHV LRP LLSA LA FM HDRD IIHRDVKPDN IL 212 T.theileri_TM35_000021280 178 RYHPH ILRLFAYFWDDMN IFLVLEYAEGGNLQMLLE----SREN-QR--LLEDEVRV ILRPLLLALTFLH ERD IIHRDVKP EN IL 255 T.vivax_TvY486_0900240 134 RYNEN ILRLFAYFWDEER IILVLEYANGGSLLDLLN----KRKQ -QQ PC IPD EEV SN ILRQ LLLA LT FLH ERD IIHRDVKPDN IL 213 AUK3 B.ayalai_Baya_064_0240 450 RHHPNVLKLYAYFWDNER IFIVLEYAEGGDLHTLRQ ----TCPG-RR-- FQ ESEVAN IIRQ LTAA LA FLHQ HG ILHRD LKP ENV L 527 P.confusum_PCON_0051580 97 QHCPHVLRLLAYFWDADRVILVMDFAEGGDLKQWLE----RRRA-AAAPVTEVEAAT IVRQ LAAALAFIH A R H IV H R D IK P EN V L 176 B.saltans_BSAL_23410 122 -KHRN IVRLLAYFWDSERVFLVLDYADEGNLATLLQ----AVPQGQR--LSEWAVRSLAGQ LCSAVLY LHRRG ILHRD IKP EN IL 199 Prokinetoplastina_PhF_6_TR32404|c0_g1_i1 274 -RHRNVLR IFAW FWDESRVYLVLEYAPGGSLSELLR------ET- IH-- FP EETVRRYTYD LLCG IRY LHTRG IVHRD IK LEN LL 348 Prokinetoplastina_PhM_4_TR2362|c0_g1_i3 148 -RHPNVLRLLAW FWDETR IFLVLEYAPHGNLAENLD---- LLPD-RR-- FP EA IAAGYVADVA SA LKY LHAR L IVHRD LK LEN LL 224 L.seymouri_Lsey_0037_0280 522 RHCSQ IAKLYTFFEDARQVYFTLEYAEEGDLASYAA----AQPS-RR--LAEEWVQRCVRQ LALALGYLHTHD IVHGAVAPKHV L 599 L.mexicana_LmxM.26.2440 351 RHCPH IVKLYAFFTDARRCYAALEYADAGDLASHLL----RQPR-QR--LPEAHAQV IVYHVALALRHLH EQ RVVHRAVTM RNV L 428 E.monterogeii_EMOLV88_260028700 603 RCCSH IVKLYASFTDARHCYTVLEFADGGDLASYLS----RQRH-QR--LSEGSVRSIVYQ LVLALRYLHKQ R IVHR SVTARNV L 680 T.brucei_TbAUK1 85 -RHKYLLRTYAYFFDETD IYLIM EPCSNGM LYTE LN----RVKC----- FAPPTAARYVAQ LA EA LLY LHQ HH ILHRD IKP EN IL 159 AUK1 B.saltans_BsAUK1_BSAL_09935 92 -RHKYLLRTYTYFYDATDVYLVM EPCSQGM LYSELS----RVKF-----FPPEQAAKY IAQ LAEALMYLHRHH ILHRD IKP EN IL 166 T.brucei_TbAUK2 140 -RHPN IVRLLSYY ITPRSVVLVLELCRGGSLLRRLQ----ATAE-RR--FDEGRATRY IRHVAQALAY LH EHG IVHRD LKPGN IL 216 AUK2 B.saltans_BsAUK2_BSAL_46390 88 -RHPH IVRM FSYYFSHESLV IVM ELCVKGSIRDKLD----RDGP-----FKEARASRYVRQTARA IQY LHANR IAHRD IK LEN IL 162

T.brucei_TbAUK3 218 FHG------EK LLLAD FSW AVRVNN SD------238 T.congolense_TcIL3000_9_260 212 FHG------Q R LLLAD FSW AVR LNRTN------232 T.cruzi_TcCLB.510349.80 142 FKA------Q AVK LAD FSW AVR LDRRV------162 T.grayi_DQ04_02681080 213 FKA------NHVKLADFSWAVRLDRRD------233 T.theileri_TM35_000021280 256 FK S------K EVK LAD FSW AVR LNQ GD------276 T.vivax_TvY486_0900240 214 FSTV--GNESRLRLADFSWAVRLNPSD------238 B.ayalai_Baya_064_0240 528 LQR------GRVKLADFSW SVRLMQ SA------548 AUK3 P.confusum_PCON_0051580 177 LRN------GRVK LSD FSW AVQ CDPH S------197 B.saltans_BSAL_23410 200 FKK------GC LK LAD FTW AVYM APT SA SSTTTA EERGRSRHPQHDSRGGGRTSLLSPAHYSAASSMHST IYT EDRR SP SPRRGA 278 Prokinetoplastina_PhF_6_TR32404|c0_g1_i1 349 LGGD SKTTHTTVK IAD FTW AVYC SK EN------375 Prokinetoplastina_PhM_4_TR2362|c0_g1_i3 225 LSL-----NGRVKVADFTWAVYTP------243 L.seymouri_Lsey_0037_0280 600 LKD----RYTTVR LGH F IHATR LP SSN------622 L.mexicana_LmxM.26.2440 429 LRC S-- EAGM TVK LGD FA SAVQ LPNGC------453 E.monterogeii_EMOLV88_260028700 681 LHRG-- EACPTAK LGD LA FAV ELPDGH------705 T.brucei_TbAUK1 160 LDH-----NNN IK LAD FGW SVHDPDN------180 AUK1 B.saltans_BsAUK1_BSAL_09935 167 LDH-----NNN IK LAD FGW SVHDPNT------187 T.brucei_TbAUK2 217 VDA-----RGVARLADFGW SKGLAGSAAAAV IT------NGYDASEV------252 AUK2 B.saltans_BsAUK2_BSAL_46390 163 LDN-----NGVAK LAD FGW ST LTG ES------183

T.brucei_TbAUK3 239 ------PRYC------RRHTVCGTLDY LAP EQ V LK--RGCTTKAD LW AVGVVAYRM LCGFLPFEMLDARDVCSCI299 T.congolense_TcIL3000_9_260 233 ------PRYR------RRYTVCGTLDY LSP EQ V LR--Q GCTTKADVW AVGA IAYR LLCGFLPFEMVGARDACSRI293 T.cruzi_TcCLB.510349.80 163 ------PQHS------RRYTLCGTMDYLAPEQISR--RGCTTKADVWALGILTYRMLCGNLPFEHLSAW ELCAR I 223 T.grayi_DQ04_02681080 234 ------PRHS------RRYTLCGTLDY LAP EQ V SK--RGCTTKADVW AVGV LTYRM LCGCLPLEHLSTW EICARI 294 T.theileri_TM35_000021280 277 ------IRYS------RRYTLCGTLDY LSP EL ISR--RGCTTKADVW SVGV LAYRM LCGQ AP FEHQ SAG ETC SR I 337 T.vivax_TvY486_0900240 239 ------LHRG------RRCTLCGTLDY IAP ELVTR--RGW DTK SDMW AVG ILAYRM ICGFLPFERLHPQDVCAKI299 AUK3 B.ayalai_Baya_064_0240 549 ------RLYSADATGDTLC----QGQRRLTMCGTLDY LAP EQ V LN--KGYDTGADMW SLGVM CY ELLCGFLPFEHVSPADTRALI621 P.confusum_PCON_0051580 198 ------RKHA------RRYTMCGTLDY LAP EQ VRQ --Q GYGVAADMW SLGV LTY ELLCGALPFERDNVRDTYASI 258 B.saltans_BSAL_23410 279 HVDYEAERRR------RRTTFCGTLDYLPPELIMQ--RAYDEAVDMWCVGATVYEM LV GRPPFETATVRETEER I 345 Prokinetoplastina_PhF_6_TR32404|c0_g1_i1 376 ------NSVRRTTLCGTLDY IAP EM LCG-- SEYDTGVDMW SVGVVM Y EM LYGHAPFESSEPRDTVRK I 435 Prokinetoplastina_PhM_4_TR2362|c0_g1_i3 244 ------ERAA------MRRTFCGTLDY LAP E IVA SPHP EYDTAVDVW SLGVVM FELLCGRAPFESDDPRDTVKK I 306 L.seymouri_Lsey_0037_0280 623 ------AESA------ETLLQEGAVGYVAPEVLCG--RERSCKSDMWALGVLTYELLCGYLPFEHVR ITQVKHLI 683 L.mexicana_LmxM.26.2440 454 ------ARW LGELG------GSHDGGRSLEYAAPEVVCG--RGW SFKSDMWALG ILVFEMVCGYHPFDHVSTAEMKRLV 518 E.monterogeii_EMOLV88_260028700 706 ------SRW LGKLE------SSH IAEESLDYAAPEV IQG--HGW SCKSDMWALGVLVYTM LW GHHPFEHVSSSEMKRLI 770 T.brucei_TbAUK1 181 ------RRKTSCGTPEYFPPEIVGR--QAYDTSADLWCLGIFCYELLVGKTPFVGKDTDQ ICKNI 237 AUK1 B.saltans_BsAUK1_BSAL_09935 188 ------RRKTSCGTPEYFPPELIAR--QVYDTTADLWCLGIFAFEMLVGKTPFAHAENDQVYAN I 244 T.brucei_TbAUK2 253 -----APRCAAGETD--ACSETEDGHGRLTVCGTLDYM PP ELLGG--TPC SYKADMW SLGA LLV E ILSGQ PP FYRT SQQ ET LQ A I 328 AUK2 B.saltans_BsAUK2_BSAL_46390 184 ------SRKTVCGTLDY LSP EM LAG--- SHT SKTDVW SLGVG LV EM ITGSPPFFNISEQETVRNI239

T.brucei_TbAUK3 300 ASGAI-RYP----S--QLSPTARHFLQGLLRVDEALRFSCQEALNHPFMRGDCNCRISN 351 T.congolense_TcIL3000_9_260 294 SIGDV-RYP----S--HLSVSARNLLEGLLCVAETTRFSCNQALSHPFIKGGNNYPRGC 345 T.cruzi_TcCLB.510349.80 224 TRGNV-QYP----T--HLSVEAQNFMESLLCVDEASRLSCEEALKHPFIVGNSCS---- 271 T.grayi_DQ04_02681080 295 AKGD I-EYP----A--D ISPEARYFMEALLCVDESTR FSCK EA LQ HP FLQ GRG SS---- 342 T.theileri_TM35_000021280 338 TRGN I-YYP----E--YLSIEAREFIAALLCVNESKR ISCM EA LQ HP F IRN ER SN---- 385 T.vivax_TvY486_0900240 300 AAGAV-HYP----P--N ISTGSRNFLEGLLRLKESERLSSHEALGHPFIRTGGRWWAAQ 351 AUK3 B.ayalai_Baya_064_0240 622 AKGAM-YVP----L--W LSPTAQ SFITSLLRVKEEERLTAEEALAHPFLRGTSPS---- 669 P.confusum_PCON_0051580 259 VAARV-SFP----SQPHLSEAVKHFVTSLLCGPEAM R LTAA EA LQ HP FLAADAPR---- 308 B.saltans_BSAL_23410 346 VRGQ F-Q FPRLIRD--Q LSWEAMDFVSRLLQ IDPARRMTAEQAARHPFVESILQAERQR 401 Prokinetoplastina_PhF_6_TR32404|c0_g1_i1 436 QDASV-VFPKG-GS--GVSEECKEIM IGLM EKDV VKRWKVSEVLMHVW FQGLE------484 Prokinetoplastina_PhM_4_TR2362|c0_g1_i3 307 QDAAL-AVP----C--YLTPECQDLIRGLLTKDRSTR LKPADV LAHPW IRNNVNTDNNR 358 L.seymouri_Lsey_0037_0280 684 CTGAV-YYP----Q--WVSAKAKSFVGSLLCVGEAGRCSAAEALRHPFISALSNA---- 731 L.mexicana_LmxM.26.2440 519 CSGAACHSP----H--ALSHTGMSFVRSLVCVDEAARSSAATALTHPFLSG IAAA---- 567 E.monterogeii_EMOLV88_260028700 771 CSGAVPYSA----H--SFSHTCMSFVRSLLCLHEAARSTAAAALEHPFLADGVHASTAA 823 T.brucei_TbAUK1 238 HSMHF-KIP----E--NIPSEAKDLIANLLLRDGSRR LA LHRVVNHQ FLLKYY------283 AUK1 B.saltans_BsAUK1_BSAL_09935 245 KRGTF-DMP----T--SVPEEAKDFINALLVKTGSERLALHRVVQHPFLVKYY------290 AUK2 T.brucei_TbAUK2 329 QDEGP-QLDVD-GS--VLSPLARDLALQLLQKDPNVRPTAAEVLQHPW LRGKR------377 B.saltans_BsAUK2_BSAL_46390 240 KEAQP-SLP----T--TLSLELKSLILEMLQKDPDQ R IS IDDV LANPW VNHDK------285

Figure S3. Comparison of the kinase domain of AUK1, AUK2, and AUK3 in kinetoplastids Figure S3. Comparison of the kinase domain of AUK1, AUK2, and AUK3 in kinetoplastids Multiple sequence alignment of the kinase domain of Aurora kinases in kinetoplastids with emphasis on AUK3. Putative AUK3 homologs are found even in some Prokinetoplastina.

23 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424754; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

T.brucei_Tb927.11.340 1 MTMWFPAETVPSS------ARKEGVTPRD------23 T.congolense_TcIL3000.11.280 1 MSLLNPDTTVFS------CHFGATPVR------21 T.vivax_TvY486_1100200 1 M--WGPMDEILS------LQMYGRQPLPG------21 T.cruzi_TcCLB.504109.10 1 M--AAREGSVCL------CG--GRRSHD------18 T.grayi_DQ04_01601130 1 M--WSPTAEVPL------PSNQGHWTQH------20 T.theileri_TM35_000054450 1 M--WGQTEDISG------FSYQGQHQQQ------20 B.ayalai_Baya_001_1220 1 M--LDHRSQT IASAGQPDR INRNW ESM IQQRQAY P------SRFVDV-PQM SHVP S IGYT SSPPQ RR------ENCVTHGT SSL---VHAQD 74 L.mexicana_LmxM.25.0280 1 M--SGNWS------FSSASTPPRRR------RHSPSSV------24 E.monterogeii_EMOLV88_250007800 1 M--SGRWT------FSNVSTPPQRT------GQ LSSSF------24 L.seymouri_Lsey_0027_0230 1 M--PTNSP------FSNASTPPQRK------ARMPAPF------24 P.confusum_PCON_0071700 1 M--QAAHAYQVRQ----DELRQTMRQLEACRVAYAADVSRKGLAAT IGVSAGLFDVRTGEERRWCGADCHPTGPRERRSAASTPRSPRGTGGW SHWPASSGFG G R S S IQ Q S F 106 B.saltans_BSAL_22535 1 M--DSEFASRIAS------LERSIGAVSSDRERYLSS------AVSSA------FTRGAHQQQNSGGGGFRVTFPSNGSSGG---VGTSF 67

T.brucei_Tb927.11.340 24 ------NGGLLSIGCDCAC------36 T.congolense_TcIL3000.11.280 22 ------KELHNRENCECAH------34 T.vivax_TvY486_1100200 22 ------QDGQGRW SCDPTS------34 T.cruzi_TcCLB.504109.10 19 ------AETVFAGGSECPN------31 T.grayi_DQ04_01601130 21 ------GHTNQ SQSCDRTN------33 T.theileri_TM35_000054450 21 ------QQQQQQRYLQRTNDDDNT------38 B.ayalai_Baya_001_1220 75 GLLDAFDDPDR------SATTWGNVDTDCSTTS------101 L.mexicana_LmxM.25.0280 ------E.monterogeii_EMOLV88_250007800 ------L.seymouri_Lsey_0027_0230 ------P.confusum_PCON_0071700 107 SDNDN IDDDDDD SN EDDDDDDDDDDDDGVGQ V LLSADG SHQ R ESAMM TTTTTTTTTQ LM ATRVT ST STTVTP IKVPRAGFCGTGYHGDTYGHSSDDGESRVHAEVEVDGCAR 218 B.saltans_BSAL_22535 68 ASSNATDDNRNPNNKHYDPS------AVAQHSSLRNQRPGAASSH------106

T.brucei_Tb927.11.340 37 ------HSPPR------41 T.congolense_TcIL3000.11.280 35 ------HSPAH------39 T.vivax_TvY486_1100200 35 ------LSPQR------39 T.cruzi_TcCLB.504109.10 32 ------SPP ID------36 T.grayi_DQ04_01601130 34 ------YSPYK------38 T.theileri_TM35_000054450 39 ------MCPSLN------44 B.ayalai_Baya_001_1220 102 ------NTPMRLD-R-SSYT------113 L.mexicana_LmxM.25.0280 25 ------VAASRVDV------32 E.monterogeii_EMOLV88_250007800 25 ------ASPSRVEA------32 L.seymouri_Lsey_0027_0230 25 ------ASTSRVDL------32 P.confusum_PCON_0071700 219 CAG IRYAPASPPRTFVTADSRRQRCTPTWARSESCGPSVSPPSSHAATLCAPTGVAWPASPSSARD IAVALFPRGRDDDDLEDGSGGDYRGDRVEAVTRELPRATHLRASSR 330 B.saltans_BSAL_22535 107 ------QTPPDYGLQQPSY------119

T.brucei_Tb927.11.340 42 ------DFSH ILESSHVTFANSSRGVSYK 64 T.congolense_TcIL3000.11.280 40 ------DFSH ILESPHVTFSSSSNGLCPP 62 T.vivax_TvY486_1100200 40 ------DFDCVLAPSC IAHANTGRRSQSA 62 T.cruzi_TcCLB.504109.10 37 ------DFTHVLRSAHVNFGHVSPGASSI 59 T.grayi_DQ04_01601130 39 ------EFSH ILEASH ITFAGKNAVTQ-- 59 T.theileri_TM35_000054450 45 ------DFSH IIEAAH ITLSTTG IAAQ SF 67 B.ayalai_Baya_001_1220 114 ------ISYEAHHQRGHLNADENRNSMGTI137 L.mexicana_LmxM.25.0280 33 ------LFEPPVRASFIEDA-GLYDAGDC 54 E.monterogeii_EMOLV88_250007800 33 ------LFDAQPGTSFIEDT-SLLDASAD 54 L.seymouri_Lsey_0027_0230 33 ------LYDHQPQTSFVDSTPSEKQASGY 55 P.confusum_PCON_0071700 331 GAPRASLAATGT IHPVAATVAHDMAALPGQVTMRDVLR IAAEAVAADD ICGEAAAPQWPGAAELHTPFPSVVPCESRSVDASTRGVKENTSSSSAGATHTEEA----GAPAE 438 B.saltans_BSAL_22535 120 ------AAASVAATQHLYEAAPFMHSQ-- 140

T.brucei_Tb927.11.340 65 GI-DPPVRMRSSIGSGARTQDLQVSSTSRRKRAKTSNNRGALHGVEESYLQEIDHLQQLAKQFDEERSEAVDRAEEMRAAYERSRQQ SILKEREIQ SLGDQM HRLQDKLHRA 175 T.congolense_TcIL3000.11.280 63 GA-ESC ISGQPPCCSSDSVPRLQADSVRCQMCAQ ISGARRP FQ VA EV SY LK E IGN LRG LAKQ FD SER SEA IDQ A ET LRRAY ERQ RQQ SV LK ER EFQ LLEDKVHRLQDKLQRT 173 T.vivax_TvY486_1100200 63 AI-GSPLRERPLAGVASLVQSLQVPGMDCRMCAQISSVR ESLRNV EESY LR E IEQ LQQ LV SQ FEV ERRNALKQVEASHAAFGRSREMNMVKDREIQALGGQVR QLEERLQRA 173 T.cruzi_TcCLB.504109.10 60 TA-SGTVREQPSP IVER ITQGLLASSLGCARCAQ ID G IR E S LK C V EA SY LG E IAQ LR RM V GQ ID G ERM D AVNRM EQTLLTFERGRQQ SLSKDCEIQ SLIGQ LQ QM EDRSQRL 170 T.grayi_DQ04_01601130 60 ---CTSLSDHPSPVVDDVTQGLLTPRQGCSRCAQ IN G IR E S LK G V EA SY L E E IA H LK R L IAQ V D A ER V D AANRAERALQAFDRSRQHNLTKDREIQ ELVGQ LR QTEDKLQRS 168 T.theileri_TM35_000054450 68 NVLNFSASDQPSLSVDSVTEGLLTPRKGCSRCAHLG S IR ET LK SV ESSY LEE ISQ LKRM INQ ID SERVDAVNRSEK ILSGFDRCRQQ ILSKDHEIQVLRGQ L RQ V EDN LQ R S 179 B.ayalai_Baya_001_1220 138 TGKSCVKEESPR---SEVTTSLPPSAFVCPRCRHVDH IRDVLKSAESAYLEEIAQ LRSMVAQ IDLERVAAANHSEQLQRSLEQLQKLNLEKEREVQAFRYEALLQADLLHRL 246 L.mexicana_LmxM.25.0280 55 DM EDATMM PR----- ETW AKND SGY SHQ CR SCQ DTHRLRNSLKRVEAEYVAEIESLRLQLQEATAQRTAAVRERNELLASADM SLTH ILQMEQDAQ SLRM EVVQ V SQ EK EQ L 161 E.monterogeii_EMOLV88_250007800 55 NGMQCAVALE-----GTPSEEDN IPLRQCQ SCQDTYRLRNSLKRVEEQYVSEIESLRLELQEAVAQRGLAVK ERND LLT SADTD LARR LQ A EQ EARK IY IEM RN ISSERKAL 161 L.seymouri_Lsey_0027_0230 56 FGY S------PAPQ RG EENTGVQ CW GCRQ TQ R LRT SLKR IELEY LAQ IESLQQQ LKRATAQ RA LA LQ ERD ELLTN SDAAAKQQ SM IEQ EAQ SLRV EVMQAMQERHEL 156 P.confusum_PCON_0071700 439 ASYATTAGSSGRLTVDELCLQVPPRSSSCGRCAGLRHLRESLRGVETAFRDEIQQ LRYAVSRGAAERQ DLTLLIQRHVSALDSERMAHDDTQRVAQTVSSLL RQM EW DYCAA 550 B.saltans_BSAL_22535 141 ---QQHYGANDFVVVDPYSEAF----SVCPRCAHLSKLEESLSAVDDQYRAELEHLRKMVAQVDADRLDVVQRLKSSHDDVEVLRSQVLEKERQ SSEWRQRAVVAEEELSAA 245

T.brucei_Tb927.11.340 176 EYQ LT SVQ S ICRRDA EFHR EQQ LLLFEEN ERNTR EALVFAEA----SWRTV IQ ERCSNTM S------A LAHKQ Y SSV ERH SE- ELG------250 T.congolense_TcIL3000.11.280 174 ECQ LA SVQ S ICRRDAR FHRDQ ELLLFEESFHNAR EA L ISM EA---- SW RM V LY ERW AH STC SPPVG-GHSPRGTRCSGRSVT-PCS------253 T.vivax_TvY486_1100200 174 EY EM SSVRR IN SNDVA FYHQ K EMM LFG ESESNAR EAVT SA EA----AWW CV LM EKW ANTT SAAGKG- SVSPVDGFRSGS----PLP------250 T.cruzi_TcCLB.504109.10 171 EY ELD SLRQ LYRR E IA FYHQ R EM FLFLEN ERCARAALSSTED----GWRSALETRHLSVVEVDGK IHRSYPHQ N FL---- FL-D FS------247 T.grayi_DQ04_01601130 169 EY ELA SLRHRYAND IA FYHQ R ESFFFT ET ERCGRVALTSMES----GWRTALQAKYVSIVEMG----RREQHRSFVSHCELP-DSP------245 T.theileri_TM35_000054450 180 EY E IA SM RH LHM RDVA FFNQ R E ILFFSEN EQ YARAA ILSLEA----NCW SEIFTKFMNEWS------P IQKM EGLSLSCRWQ-EVPGVSLI------259 B.ayalai_Baya_001_1220 247 QYEYDSLKESCVRDRELSHASMGLYFFSQEQSCRGELKAMET----QGRLLLQ------LLAGKEELERLRRAN-VFS------VVSQP IV--- 320 L.mexicana_LmxM.25.0280 162 RRRVQ ELEKQ SAVASERDARLGRVAVEDAEAVGRAHVESAET----SEALLLLQLCAAESARVAESVHRM PG ETRT SGAAAA------239 E.monterogeii_EMOLV88_250007800 162 QRRVLELERQGAADRNHFLQMTSILIADAETAVRRQ LEAT EK----D ENV LLRQ LRTAD FAR ISV LESLKP SSTPPRW AYATV ELP------243 L.seymouri_Lsey_0027_0230 157 RSRVHELEIFCSNSVQQQLQWKMKLLWEAEASARRDCSAAAQ----DEWRLLKELCDAESRAVKEALRLRAVATPSRCNVGA------234 P.confusum_PCON_0071700 551 KRDLAAAREECERWARM SRDAERMALLSQERDARQVIRIEAAQELCSAAMALWTSCAAAVVAAP---ALRTG SDA SRVW ARH- E IP------632 B.saltans_BSAL_22535 246 QRVHSSAQWEWERRVVFALETMMYALATTETDARLN LVAC EQ ----A SR SA LW SVADAGQ L------ELRRHKQ IEK LRK EQ ER LA-TKT LTM H EA EA EYRRAM D EA ILSGH 346

T.brucei_Tb927.11.340 251 ------VARHGC------RSEKTW NQ AYTDRPV SCPA---Q RC------TSTSRQYSGQQ EE---DC------W ATPATGM K 302 T.congolense_TcIL3000.11.280 254 ------SGRNGG------GGAAVWMHDCM ERPRSTAG---SNC------PSITR-YTSERPE---ES------PATSASAMN 304 T.vivax_TvY486_1100200 251 ------CSGRNAR------SSS----RG SV SRDR SL-----Q RC------PSITR-ESYACRE---GD------EPLDMHTIG 296 T.cruzi_TcCLB.504109.10 248 ------KENEGG------SSM----RGGLSPGLVR-----SLP------PSITR-DARKNRN---GG------GGSKASD IK 292 T.grayi_DQ04_01601130 246 ------RGSVGL------LST----CCGLSPSPMH-----QRS------PSITR-RIEPRTS---EE------GGNAM STLK 290 T.theileri_TM35_000054450 260 ------GSNNSSG IE------EHSSF----GGG LSP SPVQ ----- IR S------PSITR-YAETTRV---NN------M GV LKPA FK 309 B.ayalai_Baya_001_1220 321 ------SGGS------SSVVTPNDGYLSPSAFLPS--YRRSS------TSYPSISSRSSRLHPR---EKARAHLPPSPDSSGAK 381 L.mexicana_LmxM.25.0280 240 ------GGRCGSSCANVSGGFSPESASPASSEESPHRRTCSPRPSQ-----RRS------LTVPVDTPRLSLYPYSPPGHSGAE---SAATTSRLP 315 E.monterogeii_EMOLV88_250007800 244 ------VADRSYTCRNRSSDFSSGYSSPGPSDEGRGRRTCSPRHAQ-----KRA------LSAPVEVQRNLCASES---NDGAH---YAATATRC I 316 L.seymouri_Lsey_0027_0230 235 ------GTDSGGST ILCSTFISNYTSTPGPSDGGHERRLYSPRPSQ-----HRE------LSTPRLTRRPSV-----CGDSSRS---SSEGTTVVA 305 P.confusum_PCON_0071700 633 ------VAAQVASPRREPGSSHRSSYSKDSTYVVCTVSRPTS------MATTPVSSPRRSTTPLS---DE------RRVRGLRHS 696 B.saltans_BSAL_22535 347 QQQQRGGGM EANSGGGGGRPRAVLTAVSSTLSSALLTQ SRNVM SVDPPN-----SRSNSLHPNTKKAAPQQQ PAAAVVAPQ LLHRC LSDPH LP IGA ELEA LDDRDSNSPELA 453

T.brucei_Tb927.11.340 303 ST LLA ELHQ SKKR-----AD ET IEQ LKR ELEEK------EEV IASLRNQ LNLQEEGLQKVPRAGGSDESLMMEMQA 367 T.congolense_TcIL3000.11.280 305 SG ISLQ LQQ SKR S-----AD ET IKK LQ RD LDDK------ND IINSLQDRLANLEAEMDGLVSSRKVDESLVVDMQA 369 T.vivax_TvY486_1100200 297 GGRFVEQHQPTKR-----SDELHLKMRRELESK------NKLIDNLKQQ LSEKEKM LKQCSQ-NSFDETLVVEMQE 360 T.cruzi_TcCLB.504109.10 293 GGLLTELWQ SKRT-----LNELNEKLQRELRAR------EDQ ISTLRQQ LSVREESLVQ SSEERAEEDN LKR EMQ E 357 T.grayi_DQ04_01601130 291 GGLLTELRQ SKKV-----VDELNEKLKLELGAK------EEQ IKSLQQQLD IRDEGFLQLSREHATDAELAGEMQ E 355 T.theileri_TM35_000054450 310 GGLLTELRESKRN-----ADELNEKLQCELTKK------ETLISNLKHQ LSIREESIRRLSQESTEGEKLNKEMQE 374 B.ayalai_Baya_001_1220 382 DSLLAQLKASNED-----LREQVEALNNQVRHS------Q SALAEESLRRSSEALREERA LTA EMQ L 437 L.mexicana_LmxM.25.0280 316 CGFDAAVAATTAAAGDDSPRER IKRLECDL------ERQRRLHEMQLADERA LT SEMQ A 368 E.monterogeii_EMOLV88_250007800 317 PEIAPVLAGAQAAVIDDSAGVRVKKLEWSL------EQQRSSYEVQ LAEERA LT SEMQ E 369 L.seymouri_Lsey_0027_0230 306 CTLLPTHAAEEAA------EKVKMM EWRL------EQQRSSFEMQLADERG LTADMQ A 351 P.confusum_PCON_0071700 697 VSLANSPRRGAAP-----ADPRVLQLEHEL------DRVRELLALKEAECARLGLCLEEER SLA EEMQ D 754 B.saltans_BSAL_22535 454 AVLAAKDDEEAAARQRFTDDVSFGDLELEEDDTLGVSNLKPSLLLGVAGGAGGSESKPSATSNNDNAGNLTSISPSSELQRWQKLLAESDNARRQLEGHLLTEQ H F SN E LQ F 565

T.brucei_Tb927.11.340 368 EIDDLLLNELLLTHSCERYALETEAMEELTYA----LRW LLQQHAFALQEAPFITPQRTPAGSKQPVLPRTPGTS-PHLVEVHSA 447 T.congolense_TcIL3000.11.280 370 EIEDMVLNELLLTHSCQRHALEAEAAEELSNC----LWWMLQQHNSIASTNQ FSTPLRTPATKRQN ILPKTPETL-PHFIENSVS 449 T.vivax_TvY486_1100200 361 EIDDMLLNELQLTQSCQRSVLEAEAAEERVQA----LQWLLQKHTTSGLERRHATPQRVLTFTSPH--PVTPETL-PRHVDLHSL 438 T.cruzi_TcCLB.504109.10 358 EIDDMLRTEFFLTQTCERHRLETEAMEGRAHL----LQWLLQRHVLFSQERNLMFLERTPEKQGLTEKVW TPGT L-P FF ISSNDD 437 T.grayi_DQ04_01601130 356 EIDDMLRTEVWLTQSCERHRLEVEALEERNHV----LQWLLRHHLASLPKKCLNPHEGTPEKQNLAPLQWTQGTFSPLVCNQGNR 436 T.theileri_TM35_000054450 375 EIDDMLRTEVLLTQKCERHSLETQAMEERLEV----LYFFIRQN-T ILPEKH IDSVLRTPEKQTLAALKLTPKFI------G 445 B.ayalai_Baya_001_1220 438 EIDDLIATELILHERATRSEVELEAADVLMSH----CLWFHQALQTEAHGR------AEGGSASTSPPLTNAVAQQ 503 L.mexicana_LmxM.25.0280 369 EMDDLIENELVLLEANGR IALERDGAEAAVDV-- --VRCLLQHLGVALRSG------QVPSEEAFRVGTTRRKATLVTCP 438 E.monterogeii_EMOLV88_250007800 370 EIDDLIENELVLLEANGRVALQREFSETVADL----LRELLAHLGDNLSAR------PRFSEEVHTASETLPRRTSNTCC 439 L.seymouri_Lsey_0027_0230 352 EVDD L IEN ELM LLESVNR LT IERDA SEACT ELAAHHM LILTQHLWPEAQDN------AAVAPAVGAAKASTHADVLDLVEES 427 P.confusum_PCON_0071700 755 EMDDMVRSAHCDEEREVRGALERDERDARQDL-- --WAALVRQW------HRLPA------PYTSYLSASIRHGAANL 816 B.saltans_BSAL_22535 566 EVDD LSRRD EEST EAYRRAKM E ISELQ FR LELA------LQSHRRLEHRV------AALM T S S S S SP L SA ST A T A 628

24 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424754; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

T.brucei_Tb927.11.340 448 ASPGCN------DVTTSVAEMRAIVGDTVLEV I--KKELQGCLLP IR------486 T.congolense_TcIL3000.11.280 450 GSPRFH------ALGSSVSDVCAMVSDTV LETM --RK ELQ SH LLP LK------488 T.vivax_TvY486_1100200 439 GSQRGR------GLSVLHGELQGIIKDAVM ETL--KDGLQ SHLLPLK------477 T.cruzi_TcCLB.504109.10 438 VKPDVYVNRSKYSLSCQQQQQQQQQQPDELYAVVSQAVSETL--Q SVLTHQPFLIK------491 T.grayi_DQ04_01601130 437 MGPLMSRN------TDEESNEGLRAVVSQAVTETL--QKVLQAQ LLPLK------477 T.theileri_TM35_000054450 446 TPPNLFNGRSM IESSSQC---SGNGLSEDVA ILVSRTV EDTV--Q KVMQ GH LLP LK------496 B.ayalai_Baya_001_1220 504 PGP IGSALVTGYPDS---Q IICSNQ LVNEVEATVDRVM------ESHLQSLREYLG------550 L.mexicana_LmxM.25.0280 439 EAS------DGGACEGPVRPTTNQ------TSLGPGRLGDEL------468 E.monterogeii_EMOLV88_250007800 440 GTE------MA ISCKDEVCPTPKA------VPAMPAETGETS------469 L.seymouri_Lsey_0027_0230 428 ESA------AWR IEMSEVMYNMQQQ LYQ LL--SASRPPRADPEE------463 P.confusum_PCON_0071700 817 QPPSSHDAAAELPSG------IGWPTATNIALVPQSSPVDLR------G------APS 856 B.saltans_BSAL_22535 629 SPP SQQ DDV L------LLEK ELKV LRQ LYTAAKEDSIAARKELQN IELSRKESFLSGVTAPPALHKT LRPD EQ VDDDD EHP LGKM VT SFVVPT ST LANSTAAPS 726

T.brucei_Tb927.11.340 487 ------LSVESMRASSEELIGDL------RKAVRDAGYNAPQ LTPSI------521 T.congolense_TcIL3000.11.280 489 ------LSVESMRASSEELARNA------ENTLRAARAD ISLSSPAV------523 T.vivax_TvY486_1100200 478 ------LSVDSLRAHSEELVGRV------REGM EMAKSSAT--TPLID------511 T.cruzi_TcCLB.504109.10 492 ------RTAEA ICATEDELHKNS------NNFVQKMHYDED--DNV I------524 T.grayi_DQ04_01601130 478 ------LSVESIRTRSDELHASM------KKAVEVMHHGTE--PPLT------510 T.theileri_TM35_000054450 497 ------LSVESIRASSDELHRTM------RKAVEDSYHHED--PSKK------529 B.ayalai_Baya_001_1220 551 ------CMCQ SCSKSSEEIEAKLMT I------Q STC ITGQ A STVKQ VNDAM A LVVP------594 L.mexicana_LmxM.25.0280 469 ------IGREEMMTGALGDVDET------RRDVHNLRDD IARCHAAVG------504 E.monterogeii_EMOLV88_250007800 470 ------VACTALLADFTSVLDET------RKGVCDVGETVTVCQGALG------505 L.seymouri_Lsey_0027_0230 464 ------AW EETVKDCVREEAEEI------KEHLAALLEASA--QARVDGDALRM EM K EH F S 510 P.confusum_PCON_0071700 857 ------AVPLPGEPPTAAELGEVA------LGAVASAHQPEN------886 B.saltans_BSAL_22535 727 GVTTVVPPVA IAAPPPTAKM IAEEVLRM LSPQ FSLM SDSLAH ITKQ LSVSDDFLVPALSGGSKDHGG IDNSTKDEGGAPTLESVM ETSPLAAPSATTT--RKQPQSSFA 833

T.brucei_Tb927.11.340 522 --TMGEIKSEHAALLRN I-QRSLDDMN------545 T.congolense_TcIL3000.11.280 524 --TLDD IRAAHTSLKDFI-QQNLSGLG------547 T.vivax_TvY486_1100200 512 --ALDTLKREQAVFMDDV-KRLFSEFGAG------537 T.cruzi_TcCLB.504109.10 525 --NAAG INASHEKLLGDV-RRVLQEFC------548 T.grayi_DQ04_01601130 511 -- IADEMKNEHTNFLSDT-RQM LRELI------534 T.theileri_TM35_000054450 530 -- IIEELKKEFGGFITDI-KDILQSYT------553 B.ayalai_Baya_001_1220 595 --LAEKLSEAMPH IVDETCRQ LLDQHKSL------621 L.mexicana_LmxM.25.0280 505 --DTGTTQ ERM EALL----CRVLVEVAG------526 E.monterogeii_EMOLV88_250007800 506 --KVGTQQDRM ESLL----RQVLERLEY------527 L.seymouri_Lsey_0027_0230 511 --EVEERQCRQ EAFMVAS-STRLPQKEGA------536 P.confusum_PCON_0071700 887 ------LPSHLPAYQRHAFDDDDGGD------906 B.saltans_BSAL_22535 834 SLLLERVQNAAQAHQ SAV-RNM LESSMHGTDVR ITELSEQIRSSLRPTPNVSLMETSTTIPVGGDRGTHHGGGGVAGLATSLGSVTPVDYLSQNSTPRVLVPALPPPQQ 941

T.brucei_Tb927.11.340 546 ------TTVAPP-LCTE------LKQ LRETVVKVGRLTAHNNKVMDDKLSDILHCQS-AV------SRQM ILS 598 T.congolense_TcIL3000.11.280 548 ------SA IRSY-DPAE------LNRLREAVVKLGRLSAHNSK IMDDKLND ILNCQ S-AV------SRHVV LS 600 T.vivax_TvY486_1100200 538 ------SATTATA-AAQ E------LSQLRQTFVKMARLTTRNDK IMDDKLND ILLCQ S-AA------ARQVVLA 591 T.cruzi_TcCLB.504109.10 549 ------AANPPQ-EIVD------LSPV IDALKKVARLTSHNAKVMDDKLNDMLILQS-AV------ARELALL 601 T.grayi_DQ04_01601130 535 ------ASMDPA-KAGD------VATLRGALQKVARLTSHNSK IMDDKLND ILACQ S-AV------ATQ LAMA 587 T.theileri_TM35_000054450 554 ------T IVNTKDEKVE------IANLRSTLKKVARLMSHNAKVMDDKLNDILLCQS-AV------ARQRAVS 607 B.ayalai_Baya_001_1220 622 ------YRLNLV-QRTD------LSSVKEALKK IAHISSRNDAV IDAKLDT ILQRQ ERCL------A PHYLLA 675 L.mexicana_LmxM.25.0280 527 ------QRRK------ADETHTM LRRFAR ISTHNDNALFRAVGRVEETLK-TP------TPAAALV 573 E.monterogeii_EMOLV88_250007800 528 ------QNSQ------AEETCAALRHFSR ISTHNDDALFRAVGKVEAAVK-AQ------SH SAV-- 572 L.seymouri_Lsey_0027_0230 537 ------PTLQ------LESLRSELKQQRQ LS------ERLLEVLQCAS-AP------ASAVVTV 575 P.confusum_PCON_0071700 907 ------SGGYGVSDGAGE------LAGGRSR-HDSSRSTPRHDRGYA ITPLDVARTEDLLQ------QSFTSIS 961 B.saltans_BSAL_22535 942 QQGSGGNSSRDQ SQ IIIPHSLSSPLAPGGQAPQQQNDPVALSKLQDA IASMRN IMRNVLFSTSHNQK LIGEKLDDLTIGQR-ALMNEWNTTAASLLDSSQRHALQLSAF 1049

T.brucei_Tb927.11.340 599 ALPCDEKLDFCRTPVAEC------STTVPPAK------VSLSREGANA ISSVGGGLNYNAEN------648 T.congolense_TcIL3000.11.280 601 SLPCGEGLGTARTPETAS------PTAAGNTK------GST------629 T.vivax_TvY486_1100200 592 TM PPVGNPR SAN SSHAN S------SH IASPSK------LST IGTSANLSFSSADDQRTPSLN------641 T.cruzi_TcCLB.504109.10 602 TTPSAGR-RVDESPSSNV------SATAS------LSIVDAGVAGANNKASP------640 T.grayi_DQ04_01601130 588 AVPH IEE-ELHDAPPLEI------SAAVSPCK------REGD--- ISSTAAAASAGSGKASC------633 T.theileri_TM35_000054450 608 DV ISSVEGKVYD IEPLNA------SSTLTSSG------VE-----VSTTVSPVPN IEKTVSP------652 B.ayalai_Baya_001_1220 676 QQ ESLPNPAEACSSAAKM EKHVEDTARRTDDGKNCTTP------RNSSAGSPTD------LTPETASSPERSCSKGPAEKATDG------747 L.mexicana_LmxM.25.0280 574 KSGE----AGRGASAAKC------TAVLDSPSP------LGAEVVPCMRAMDVTRSTTTTDES------620 E.monterogeii_EMOLV88_250007800 573 KTGD I---ETDGQWGSRH------GSIYAFPSK------ELRTEGRGSEAAAD I-HPPNSTKHS------620 L.seymouri_Lsey_0027_0230 576 AAHP----SSSTTSSTEM------RPSSHNPKP------GPTSRLGASLPDTV-TPKRKTRHR------621 P.confusum_PCON_0071700 962 SMPTPSWKSPSPSAAPER------SRSRSPLP------LGSAVAVVDGEPGDPDGGNR------1007 B.saltans_BSAL_22535 1050 SPPH SG SG SHGGQ SAVMM ---G SPTYV SPTD SVNM GSPTMQHHHHGSGSQNTGPSPILQHNGHSFRQNGGGGSVQQQQQQ LMPSIAAAQNESNNNTATSAVQYSVPPA L 1155

T.brucei_Tb927.11.340 649 ------VLSDGGNESGVSI------661 T.congolense_TcIL3000.11.280 630 ------RKDESELGVSH------640 T.vivax_TvY486_1100200 642 ------NSRQVRF------648 T.cruzi_TcCLB.504109.10 641 ------ISRRAHPKPLN------651 T.grayi_DQ04_01601130 634 ------ESKQVRFELAE------644 T.theileri_TM35_000054450 653 ------KSKHVRFDLTH------663 B.ayalai_Baya_001_1220 748 ------THKQVRFHVTPETVKQVTL 766 L.mexicana_LmxM.25.0280 621 ------AADVSNDLR------Q LPSRS------635 E.monterogeii_EMOLV88_250007800 621 ------DATLLPSVP------ASPSYSV------636 L.seymouri_Lsey_0027_0230 622 ------DPLA------EKSTMPQAPH------AAPSKSSEFGHHNHK------650 P.confusum_PCON_0071700 1008 ------APPTLPVW SG IHVKK------1022 B.saltans_BSAL_22535 1156 QQKALAALAAQNAAVNRPPPQALHPTHLQN ITDQQHQEATHVIDQFVLEEAQQQQQLFQQQQQYHPGSGGHYVTSPAAPPLPQ FSKVTSTPAQQHH IQQQQQ YYQQQQH 1264

T.brucei_Tb927.11.340 662 ------KKTQ SHESSGAYV------DSTGPAFHDWDVESSDSK------FMVPSMDVSIIR 704 T.congolense_TcIL3000.11.280 641 ------GQCEKGGDKIRQPFNNSGTGS------HLISAAFHDWDVESACSG------LTALSVDASAVR 691 T.vivax_TvY486_1100200 649 ------ADEKLMKDKVSNARV------ESTSPVCRDVARATDSRR------TTMLAPAESAKR 693 T.cruzi_TcCLB.504109.10 652 ------LDKSPAGRGEGNAPV------ILQAKPSHEWDVAESDDE------SIPPIP V F S S LR 696 T.grayi_DQ04_01601130 645 ------DDAQ SKGRGVSGASF------SGSTLPSHVWDVVESDDE------SIPPPPTAAVTR 689 T.theileri_TM35_000054450 664 ------YDSSLTGEKHDNTLF------NTSVPLSHEWDVEVSDDE------TVPSPPASSIKR 708 B.ayalai_Baya_001_1220 767 Q----PS------PTVEAGEP IATEWNGDTRTLESD-----E-----DLSIPM DNSLLSLPAH IWDLDDSDDE------P IP V TQ G G N F 829 L.mexicana_LmxM.25.0280 636 ------PFAAAEDDLFDPSRVHVDSDVAAQKMAALL---LEDSLSDPSDGDVP 679 E.monterogeii_EMOLV88_250007800 637 ------NCVVAEEK LFDP SR IHVDVNVAAVQM AG FV---DYDTV SECASQAVS 680 L.seymouri_Lsey_0027_0230 651 -----PL------TAAGPLCEDQLSTGATVSDSTADCDPLPLRRSRIPDPRFVVAEEELFDPQC ISVDASAAAEQ ISALL---SDGSLSDAD----- 728 P.confusum_PCON_0071700 1023 ------WTPYDDDDDDDDATSRTMA------TATDSRCDSARSSGDADSRADS------PPVARQR 1070 B.saltans_BSAL_22535 1265 GVAVPPSAV IVVPSQHLGGAQHVHRSGSPAHSGDRSSSPGGGGGSSG------SGLP------PLQRRASHRWDEDEDDEG-SPVVPVSRATSSVAPAAAVAQ 1354

T.brucei_Tb927.11.340 705 K------TPYD IED-----A 713 T.congolense_TcIL3000.11.280 692 K------TPYDADD----- I 700 T.vivax_TvY486_1100200 694 R------TPYDVDD----- I 702 T.cruzi_TcCLB.504109.10 697 K------TPYDADE-----L 705 T.grayi_DQ04_01601130 690 R------TPYD IDE-----V 698 T.theileri_TM35_000054450 709 R------TPYD IDD-----A 717 B.ayalai_Baya_001_1220 830 R------TPYDDM E-----N 838 L.mexicana_LmxM.25.0280 680 Q------TPYDDY------686 E.monterogeii_EMOLV88_250007800 681 Q------TPFDIFE------688 L.seymouri_Lsey_0027_0230 729 ------GDFPSPYEEYGEALGSA 745 P.confusum_PCON_0071700 1071 RDGLLALEIGAEW IGRFSTP------S 1091 B.saltans_BSAL_22535 1355 R------SW-KKVKNPYSDSD------1368

Figure S4. Sequence analysis of TbABP79 Multiple sequence alignment of TbABP79 homologs in kinetoplastids.

25 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424754; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Supplemental Tables

Table S1: List of proteins identified in the immunoprecipitates of YFP-TbMad2 and YFP-

TbAUK3 by mass spectrometry (Excel file)

Table S2. Trypanosome cell lines used in this study

Name Description SmOxP9 Parental cell line, TREU 927/4 procyclic form cells expressing that expresses TetR and T7 RNAP (Poon et al., 2012)

BAP72 YFP-TbMad2 (Akiyoshi and Gull, 2013) BAP59 YFP-TbMBP65 (this study) BAP76 YFP-TbAUK3 (this study) BAP81 YFP-TbABP79 (this study)

Table S3. Plasmids used in this study

Name Description pEnT5-Y TY-YFP tagging vector, Hygromycin (Kelly et al., 2007) pBA2 TY-YFP-TbMad2 tagging construct, Hygromycin (this study) pBA38 TY-YFP-TbMBP65 tagging construct, Hygromycin (this study) pBA12 TY-YFP-TbAUK3 tagging construct, Hygromycin (this study) pBA54 TY-YFP-TbABP79 tagging construct, Hygromycin (this study)

Table S4. Primers used in this study

To make Primer sequence (all are listed in the 5’-to-3’ direction) pBA2 Following two PCR fragments were cloned into pEnT5-Y using XbaI and BamHI - TbMad2 CDS targeting sequence with XbaI and NotI BA1: GATCGATC TCTAGA GGAGCAGGT GCAGAGAGGTTACAAACCATTAC BA2: GATCGATC GCGGCCGC AGACCCACTTTATCACCTGC - TbMad2 5'UTR targeting sequence with NotI and BamHI BA3: GATCGATC GCGGCCGC TGTGGAGTTTATGTGATGTG BA4: GATCGATC GGATCC TTCTTGCGAGATGCAGCACC

pBA38 Following two PCR fragments were cloned into pEnT5-Y using XbaI and BamHI - TbMBP65 CDS targeting sequence with XbaI and NotI BA172: GATCGATC TCTAGA GGAGCAGGT GAATGCGCGTCGCAGGAGTT BA173: GATCGATC GCGGCCGC TCCCAACTACTGTCATGACT - TbMBP65 5'UTR targeting sequence with NotI and BamHI

26 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424754; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

BA174: GATCGATC GCGGCCGC TGTGTTTATTTTTCACATCC BA175: GATCGATC GGATCC AGGGTTGTCCAATGAAGGAG

pBA12 Following two PCR fragments were cloned into pEnT5-Y using XbaI and BamHI - TbAUK3 CDS targeting sequence with XbaI and NotI BA43: GATCGATC TCTAGA GGAGCAGGT AGCACACGTCTGTGCGAGCC BA44: GATCGATC GCGGCCGC TTCGTATTATAACAAAATCG - TbAUK3 5'UTR targeting sequence with NotI and BamHI BA45: GATCGATC GCGGCCGC AGTTGTTACTTCCTTTTACT BA46: GATCGATC GGATCC GTATGATCACTATAGCAGGC

pBA54 Following two PCR fragments were cloned into pEnT5-Y using XbaI and BamHI - TbABP79 CDS targeting sequence with XbaI and NotI BA236: GATCGATC TCTAGA GGAGCAGGT ACTATGTGGTTTCCTGCCGA BA237: GATCGATC GCGGCCGC CTTGCGTGCGGGCACCGGAA - TbABP79 5'UTR targeting sequence with NotI and BamHI BA238: GATCGATC GCGGCCGC GTGGCGGTAGTCCACATTAT BA239: GATCGATC GGATCC TTGGTAAAGGAGAGAGCACA

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