Interactions of p24 proteins characterized by yeast two-hybrid, mutagenesis, and overexpression
Elaine Tan
Department of Biochemistry McGill University Montreal, Quebec February, 2009
A thesis submitted to McGill University in partial fulfillment of the requirements for the degree of Master of Science
© Elaine Tan, 2009
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Abstract
The evolutionary conserved and abundant p24 proteins cycle through the early secretory pathway, but their cellular functions are still poorly understood.
They are not essential in yeast, but in mice, p23 knockout is embryonic lethal.
Moreover, p23 is involved in Alzheimer’s disease pathogenesis. The p24 proteins have the ability to form hetero-complexes. Here, the pairwise interactions among p24s, in yeast and human, were determined by yeast two-hybrid. The p24 interactions are specific and occur mostly through their GOLD domain with some contribution from their DOG sequence. Mutagenesis experiments suggested that two p24s interact differently with a common p24 partner. Finally, co- overexpression of p24s in yeast has a harmful effect on a subset of combinations.
Some have a growth defect whereas others have a cell size increase phenotype or both. I propose that the p24 proteins might participate in transport of GPI- anchored proteins involved cell wall maintenance or cell cycle pathways.
ii Résumé
Les abondantes protéines de la famille p24 sont conservées dans l’évolution et circulent dans la voie sécrétoire précoce, mais leurs fonctions cellulaires sont encore mal définies. Ils sont dispensables chez la levure, mais un knock-out de p23 est létal embryonnaire chez la souris. De plus, p23 est impliqué dans la pathogénèse de la maladie d’Alzheimer. Les protéines p24 peuvent former des hétéro-complexes. Dans cette étude, les interactions des p24s sont déterminées par double hybride. Elles sont spécifiques et se font surtout par leur domaine GOLD avec une contribution de leur séquence DOG. Les expériences de mutagénèse révèlent que deux p24s interagissent différemment avec un p24 commun. La co-surexpression de certains p24s dans la levure cause la mort ou l’élargissement des cellules. Je propose que les protéines p24s participent dans le transport des protéines à ancrage GPI impliquées dans l’entretien de la paroi cellulaire et dans les voies du cycle cellulaire.
iii Preface
The work presented in this thesis is essentially my own. The majority of the plasmids required to perform the yeast two-hybrid experiments were previously constructed by other members of the Thomas Laboratory unless otherwise indicated. The protocol for setting up the crystallization screen was done by Yazan Abbas from Dr. Bhushan Nagar’s Laboratory (McGill) and the
NMR experiment was done by Dr. Tara Sprules from the Québec/Eastern Canada
High Field NMR Facility (QANUC, McGill). Dr. Gregor Jansen and Dr. David
Thomas provided advice and supervision throughout the course of the studies.
This work is supported by the Fonds de la recherche en santé du Québec (FRSQ).
iv Table of Contents
Abstract ii
Résumé iii
Preface iv
Table of Contents v
List of Figures ix
List of Tables x
List of Abbreviations xi
Acknowledgements xiii
Chapter 1 Introduction 1
1.1 The secretory pathway 2
1.1.1 The endoplasmic reticulum 2
1.1.2 The Golgi apparatus 4
1.1.3 The ER-to-Golgi intermediate compartment (ERGIC) 4
1.1.4 Vesicular trafficking in the secretory pathway 5
1.1.5 COPII vesicle formation and anterograde transport 5
1.1.6 COPI vesicle formation and retrograde transport 7
1.2 The p24 proteins 8
1.2.1 Domains and motifs of p24 proteins 8
1.2.2 Yeast p24 proteins and their possible functions 9
1.2.3 Mammalian p24 proteins 11
1.2.4 Involvement of mammalian p24s in transport 13
1.2.5 Structural roles of mammalian p24 proteins 15
v 1.2.6 Alzheimer’s disease and p24 proteins 16
1.2.7 The p24 proteins of other organisms 17
1.3 Project proposal 19
Chapter 2 Material and Methods 21
2.1 Strains, cell lines, and media 22
2.2 Primers and plasmids 22
2.3 Membrane yeast two-hybrid system (MYTHS) 22
2.3.1 Construction of yeast two-hybrid plasmids 22
2.3.2 Verification of protein C-terminus orientation and 25 expression level
2.3.3 Bacterial transformation 26
2.3.4 Yeast transformation 26
2.3.5 Yeast two-hybrid experiment (MYTHS) 26
2.4 Random mutagenesis by PCR 27
2.4.1 PCR experiment to generate random mutations 27
2.4.2 In vivo recombination 27
2.4.3 Mutants screening 27
2.4.4 Plasmid DNA extraction from yeast 28
2.5 Yeast three-hybrid 28
2.5.1 Knockout by gene replacement 28
2.5.2 High efficiency yeast transformation 29
2.5.3 Yeast chromosomal DNA extraction 30
2.5.4 Yeast three-hybrid experiment 30
2.5.5 Plasmid loss 30
vi 2.6 Co-overexpression in yeast 31
2.6.1 Cloning and transformation into yeast 31
2.6.2 Serial dilution screen 31
2.6.3 Growth curves 31
2.6.4 Recovery in dextrose 32
2.6.5 Cell size assessment by microscopy 32
2.6.6 Staining with fluorescent dyes 32
2.7 Expression in insect Sf9 cells 33
2.7.1 Cloning into plasmid and conversion into bacmid 33
2.7.2 Bacmid transfection and large-scale infection of Sf9 33 insect cells 2.7.3 Purification of protein secreted into the medium by 34 infected Sf9 cells 2.8 Coexpression in bacterial cells 34
2.8.1 Cloning with dual expression vectors 34
2.8.2 Large-scale coexpression 35
2.9 Protein purification 35
2.9.1 Ni-NTA purification of His6-tagged proteins 35
2.9.2 Dialysis of Ni-NTA purified proteins 36
2.9.3 Mono Q chromatography 36
2.9.4 Gel filtration 36
2.10 SDS-PAGE and Western blotting 36
2.11 Pull-down assays 37
2.11.1 Coupling of protein of interest to beads 37
2.11.2 Rat smooth ER preparation 38
vii 2.11.3 Pull-down experiment 38
2.11.4 Silver staining 38
2.12 Crystallization screen 39
2.13 NMR experiment 39
2.14 Circular dichroism 39
Chapter 3 Results 40
3.1 Interactions of yeast p24s determined by MYTHS 41
3.2 Interactions of human p24s determined by MYTHS 51
3.3 Components of the -secretase 51
3.4 Random mutagenesis of Erp4G 55
3.5 Yeast three-hybrid of Erp1G, Erp6G, and Erp4p 58
3.6 Co-overexpression of p24s in yeast 62
3.7 Expression of recombinant p24 proteins 73
3.8 NMR, CD, crystallization screen, and pulldown assay 75
Chapter 4 Discussion 81
4.1 Interactions between yeast p24 proteins 82
4.2 Human p24s and -secretase 87
4.3 Co-overexpression of p24 proteins in yeast 89
4.4 Expression of recombinant p24 proteins 95
Conclusions 95
APPENDIX A 97
APPENDIX B 100
References 102
viii List of Figures
Chapter 3 Results Page
Figure 3.1 The principle of the membrane yeast two-hybrid system 43 (MYTHS) and the hyperactive RNAse system
Figure 3.2 Interaction maps of the luminal and GOLD domains of 45 yeast p24 proteins
Figure 3.3 Interaction maps of the DOG sequences and full length of 49 yeast p24 proteins
Figure 3.4 Interaction maps of human p24 proteins 52
Figure 3.5 Erp4G mutants generated by PCR random mutagenesis 56
Figure 3.6 Yeast three-hybrid experiment of Erp1G, Erp6G, and 60 Erp4p
Figure 3.7 Growth phenotype of W303-1A cells that co- 65 overexpressed p24 proteins.
Figure 3.8 Recovery on dextrose after induction on galactose for the 67 cells that did not grow when co-overexpressing a subset of p24s in the serial dilution screen
Figure 3.9 Cell size phenotype of W303-1A cells that co- 69 overexpressed p24 proteins.
Figure 3.10 Phenotype of co-overexpressed ERV25-ERV25 and stained 71 with Calcofluor White
Figure 3.11 Expression of yeast and human p24 proteins in Sf9 insect 77 cells
Figure 3.12 Expression tests and large-scale coexpression and 79 purification of His6-TMP21L
ix List of Tables
Page
Chapter 2 Material and Methods
Table 2.1 List of strains 23
Table 2.2 List of media 23
Table 2.3 List of plasmids 24
Chapter 3 Results
Table 3.1 Interactions between GOLD and luminal domains of yeast 48 p24 proteins
Table 3.2 Interactions of DOG sequences with GOLD and Luminal 48 domains of yeast p24 proteins
Table 3.3 Interactions of yeast p24 proteins that only depend on the 48 GOLD domain
Table 3.4 Signal sequence, number of TMD span, and C-terminus 54 orientation of -secretase components
Chapter 4 Discussion
Table 4.1 Genes involved in vacuolar function and genetic 91 interactors with p24s
Table 4.2 Genetic p24 interactors involved in cell wall maintenance 93
Table 4.3 GPI-anchored proteins involved in cell cyle or cell wall 94 maintenance
x List of Abbreviations
AAA – ATPase Associated with various cellular Activities AD – Alzheimer’s Disease AD-HSP – Autosomal Dominant Hereditary Spastic Paraplegia ADE – ADEnine AMP – AMPicillin APH-1 – Anterior Pharynx Defective 1 ARF1 – ADP-Ribosylation Factor 1 ATCC – American Type Culture Collection BiP – Binding Protein bp – base pair CD – Circular Dichroism COP – COat Protein CS – ccdB Survival CV – Column Volume DOG – Downstream of GOLD EDTA – EthyleneDiamineTetraAcetic acid ER – Endoplasmic Reticulum ERAD – ER-Associated Degradation ERGIC – ER-to-Golgi Intermediate Compartment EV – Empty Vector GAL – GALactose Gas1p – Glycosylphosphatidylinositol-Anchored Surface protein GOLD – GOLgi Dynamics GAP – GTPase Activating Protein GDP – Guanine DiPhosphate GTP – Guanine TriPhosphate GEF – Guanine nucleotide Exchange Factor HAC1 – Homologous to Akt/Creb 1 HYG – HYGromycin INO – INOsitol IPTG – IsoPropyl β-D-1-ThioGalactopyranoside IRE1 – Inositol Requiring Enzyme 1 KAN – KANamycin KAR2 – KARyogamy 2 kDa – kilo Dalton LB – Luria Bertani broth LAi - Leucine Adenine inositol LEU – LEUcine LT – Leucine Tryptophan LTAi – Leucine Tryptophan Adenine inositol LTU – Leucine Tryptophan Uracil LTUAi – Leucine Tryptophan Uracil Adenine inositol LU – Leucine Uracil MAT – MAting Type MCS – Multiple Cloning Site
xi min - minute MS – Mass Spectrometry MYTHS – Membrane Yeast Two-Hybrid System NAT – Nourseothricin NMR – Nuclear Magnetic Resonance OD600 – Optical Density measurement taken at a wavelength of 600nm PBS – Phosphate-Buffered Saline PCR – Polymerase Chain Reaction PEN-2 – Presenilin ENhancer 2 PEG – PolyEthylene Glycol PM – Plasma Membrane RT – Room Temperature rpm – Revolution Per Minute SC – Synthetic Complete SD – Synthetic Dropout SE – Sorbitol-EDTA SEC – SECretory SDS-PAGE – Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis SNARE – Soluble NSF Attachment protein REceptor SS – Signal Sequence TB – Terrific Broth TBS-T – Tris-Buffered Saline containing Tween-20 TE – Tris-EDTA TGN – Trans-Golgi Network TMD – TransMembrane Domain TMP21 – TransMembrane Protein of 21kDa TRP – TRyPtophan TTIs – Tubular Transport Intermediates TU – Tryptophan Uracil UPR – Unfolded Protein Response UPRE – Unfolded Protein Response Element URA – URAcil VTC – Vesicular Tubular Cluster WT – Wild Type XBP1 – X-box Binding Protein 1 YPD – Yeast Peptone Dextrose Y2H – Yeast 2-Hybrid Y3H – Yeast 3-Hybrid
xii Acknowledgements
I would like to thank my supervisor, Dr. David Thomas, for giving me the opportunity to do my Masters’ degree in his lab as well as his help and advice. I would also like to thank past and present members of the laboratory for their help throughout the course of these studies especially Dr. Gregor Jansen for his guidance and advice, Pekka Maattanen for his advice in protein purification and other protein experiments, and Jamie Surprenant for teaching me how to use the two robots of the Thomas Laboratory. I would like to express my thanks to people from Dr. Bhushan Nagar’s lab who have offered me their assistance: Elisa for teaching me how to do insect cell culture, Yazan Abbas, for his help in setting up the crystallization screen, Rose Szittner for her time and generosity, and Dr.
Nagar for his advice about alignments and protein modeling. I would additionally like to acknowledge members of my Research Advisory Committee, Dr. Jason
Young and Dr. Kalle Gehring, for their advice. Finally, I would like to express my appreciation to my parents, siblings, and friends for their support and understanding throughout the duration of my Masters studies.
xiii
Chapter 1
Introduction
1 1.1 The secretory pathway
Eukaryotic cells are compartmentalized into organelles of diverse environments. Each organelle performs essential activities for the proper function of the cell. The secretory pathway, which mainly consists of the endoplasmic reticulum (ER), ER-to-Golgi intermediate compartment (ERGIC), Golgi apparatus, and plasma membrane (PM), has evolved to transport newly synthesized secretory proteins to various cellular compartments by means of transport vesicles (reviewed by Spang, 2008). Secretory proteins are first synthesized in the cytosol and transported into the ER, the starting point of the secretory pathway. The polypeptides are folded inside the ER with the help of ER chaperones and are packaged into vesicles for transport through the secretory pathway until they reach their destination. Meanwhile, the identity of each organelle along the pathway is maintained by sorting and retrieval systems.
Misfolded polypeptides are subjected to quality control (reviewed by Anelli,
2008). Thus, the activities of the secretory pathway are tightly coordinated and defects can lead to diseases. The secretory pathway is highly conserved between the unicellular budding yeast Saccharomyces cerevisiae and higher eukaryotes, including the complex multicellular human being, which makes yeast a powerful tool of study. The organelles of the early secretory pathway, which include the ER,
ERGIC, and Golgi apparatus, are briefly described in sections 1.1.1 to 1.1.3.
1.1.1 The endoplasmic reticulum
The ER is an organelle continuous with the nucleus (reviewed by Lowe,
2007) and its lumen is a site where numerous cellular processes occur to ensure
2 cell integrity. Protein folding, glycosylation (reviewed by Molinari, 2007), and secretion, intracellular calcium homeostasis, and lipid biosynthesis are among those processes (reviewed by Kim, 2008). The level of calcium is important as the calcium-dependent molecular chaperones help in the stabilization of protein- folding intermediates. The ER lumen also has an oxidative function as the formation of protein disulfide bonds occurs there (reviewed by Mamathambika,
2008). Disturbances such as overexpression of proteins, inhibition of glycosylation, and calcium depletion lead to an accumulation of unfolded proteins in the ER. Cells experiencing such threats trigger the activation of the unfolded protein response (UPR, reviewed by Malhotra, 2007). Briefly, accumulation of unfolded proteins leads to the dimerization of Ire1p. This event triggers the trans- autophosphorylation of Ire1p kinase domain and the activation of its endoribonuclease domain (RNAse), which splices the intron of HAC1 mRNA in yeast (XBP1 in mammalian cells) to initiate its translation. The transcription factor Hac1p binds to unfolded protein response elements (UPRE) and activates the transcription of genes with such element. The main role of the UPR is to reestablish normal ER environment by increasing the transcription of ER chaperones, such as Kar2p (BiP in mammalian cells), to increase protein folding.
Proteins that would not attain their native conformation are degraded through the ER-associated degradation (ERAD) pathway. Some ERAD factors are transcribed under UPR conditions (reviewed by Vembar, 2008). Briefly, misfolded proteins are retrotranslocated into the cytosol through the Sec61 translocon, deglycosylated, polyubiquitinated, and then degraded by the 26S
3 proteasome. Properly folded proteins are packaged into COPII vesicles to exit the
ER and travel through the secretory pathway to their destination (Section 1.1.5).
1.1.2 The Golgi apparatus
The Golgi apparatus is made up of stacked cisternae (sac-like structures) in mammalian cells, but is dispersed throughout the cytosol in yeast (reviewed by
Lowe, 2007; Prydz, 2008). Briefly, the Golgi consists of four distinct sections termed cis-, medial-, trans-Golgi, and trans-Golgi network (TGN), where enzymes residing in a particular section are usually not found in another one.
Glycoproteins and proteoglycans undergo further modifications by proteases, glycosyltransferases, and other types of enzymes in different compartments of the
Golgi. However, transport within the Golgi is still a subject of debate and two models have been proposed (reviewed by Pelham, 2006). First, the vesicular model predicts that the Golgi stacks are stable structures and secretory proteins move through them in vesicles. Second, the cisternal maturation model predicts that cisternae are transient structures that form de novo, mature in a cis-to-trans direction, and then dissipate. Secretory proteins would follow with the maturation.
Recent data leans towards the cisternal maturation model in yeast (Matsuura,
2006; Losev, 2006). ER-resident proteins are packaged into COPI vesicles at the cis-Golgi and return to the ER in a retrograde fashion (Section 1.1.6).
1.1.3 The ER-to-Golgi intermediate compartment (ERGIC)
Another organelle found in mammalian cells, but seemingly not in yeast is the ERGIC or vesicular tubular clusters (VTCs). Briefly, the ERGIC is a membranous compartment located between the ER and the Golgi, but is not
4 continuous with either (Fan, 2003). It was first defined with the identification of
ERGIC-53 that is predominantly localized on these membranes (Schweizer, 1988).
In mammalian cells, the ERGIC is the first post-ER sorting station for anterograde transport. However, protein sorting in this compartment is bidirectional and its mechanism of action is controversial (reviewed by Appenzeller-Herzog, 2006).
1.1.4 Vesicular trafficking in the secretory pathway
Protein trafficking through the secretory pathway depends on transport vesicles (reviewed by Spang, 2008). The current model of vesicle generation is the priming complex (Springer, 1999). Briefly, several components must be present from a donor membrane in order for the vesicular budding mechanism to function: cargo that needs to be transported, a small GTPase of the ARF/SAR family, coat proteins, and SNARE proteins. Anterograde transport is mediated by
COPII vesicles budding from the ER and moving toward the Golgi apparatus.
Retrograde transport is mediated by COPI vesicles budding from the Golgi or the
ERGIC toward the ER for retrieval of ER-resident proteins. COPI vesicles might also mediate intra-Golgi transport, but this concept is still controversial (Section
1.1.2). A third type of transport vesicles is the clathrin-coated vesicles that mediate transport in the late secretory and endocytic pathways. They are present in the TGN, endosomes, and the PM (reviewed by Mills, 2007). The COPII and
COPI vesicles are further described in sections 1.1.5 and 1.1.6 respectively.
1.1.5 COPII vesicle formation and anterograde transport
Anterograde trafficking is the forward transport of newly synthesized secretory proteins from the ER to the Golgi and is mediated by COPII vesicles.
5 This process is balanced by retrograde transport with which proteins that have escaped from the ER or that have accompanied the cargo as adaptors or receptors are retrieved (Section 1.1.6). The COPII vesicles are probably the best characterized of all vesicles as the crystal structures of all basic components and complexes are solved (Stagg, 2006; Fath, 2007). COPII vesicle formation, which can only be initiated at the ER, is a sequential process and starts with the recruitment of the small GTPase Sar1p to the ER membrane by the nucleotide exchange factor (GEF) Sec12p, which is a type II transmembrane protein. Upon exchange from GDP to GTP, Sar1p undergoes a conformational change whereby it exposes it amphipathic N-terminal tail and inserts it into the ER membrane.
Then, the GTPase activating protein (GAP) Sec23p and the cargo recruiter
Sec24p bind to Sar1p and cargo to form a complex at the ER membrane.
Sec13p/Sec31p complex is recruited and binds to membrane and Sec23p/Sec24p complex to help in deforming the ER membrane and stabilizing the polymerizing coat. Other components serve to help the process of COPII formation. For example, Sec16p is not found in the vesicles, but is thought to serve as a scaffold to coordinate their assembly. In mammalian cells, COPII vesicles only form at the transitional ER (tER) or ER exit sites (ERES) whereas they seem to form everywhere along the ER membrane in yeast cells (reviewed by Hughes, 2008).
After pinching off the ER membrane, the subsequent steps such as vesicle docking and fusion to target membrane require numerous proteins such as tethers,
SNAREs, and Rab GTPases (reviewed by Cai, 2007). In yeast, COPII vesicles individually fuse with the cis-Golgi, but in mammalian cells, they undergo homotypic fusion with each other to generate ERGIC clusters. This fusion event is
6 required for concentration of cargo proteins into fewer, but larger transport carriers, thereby decreasing traffic congestion between ER and Golgi and rendering the transport process more energy efficient. At the ERGIC, escaped proteins return to ER by means of COPI vesicles.
Some cargo proteins need a specific adaptor or receptor to help their incorporation into the COPII vesicles. Examples of adaptors include Erv14p
(Powers, 2002) and Erv29p (Belden, 2001c), which are required for the transport of the transmembrane protein Axl2p and the soluble glycosylated pro-α-factor
(gpαf) respectively. Another example is the family of p24 proteins, whose putative role is to serve as adaptors or receptors for the transport of subset of secretory proteins (reviewed by Kaiser, 2000; Section 1.2).
1.1.6 COPI vesicle formation and retrograde transport
Retrograde trafficking is the transport from the Golgi towards the ER and is mediated by COPI vesicles (reviewed by Bethune, 2006b). COPI formation can occur at different membranes along the secretory pathway including Golgi and
ERGIC, unlike COPII (Section 1.1.5). The vesicle generation requires the activation of the small GTPase Arf1p by a GEF of the Sec7p family (reviewed by
Casanova, 2007). Upon nucleotide exchange from GDP to GTP, Arf1p-GTP undergoes a conformational change and inserts its exposed N-terminal amphipathic helix into the Golgi membrane. Coatomer, a coat protein complex that comprises seven subunits (Ret1p, Sec26p, Sec27p, Sec21p, Ret2p, Sec28p, and Ret3p in yeast; α, β, β’, γ, δ, ε, and ζ in mammalian cells, respectively) is recruited to the membrane followed by ArfGAP1p. The coatomer is the main
7 cargo recruiter and acts to deform the membrane. After pinching off the membrane, the COPI vesicles are uncoated by triggering GTP hydrolysis by
Arf1p whose activity is enhanced by ArfGAP1p. The delivery of cargo to target organelle includes events such as docking and fusion, which are mediated by tethering factors and SNARE proteins respectively (reviewed by Cai, 2007).
In mammalian cells, the p24 proteins are involved in COPI vesicle biogenesis and regulation (Section 1.2.4).
1.2 The p24 proteins
1.2.1 Domains and motifs of p24 proteins
The p24 proteins are abundant in the early secretory pathway and are conserved throughout evolution. They are type I transmembrane proteins of about
23-27kDa. They have a small C-terminal cytoplasmic tail of about 10-15 amino acids and a larger luminal domain, which comprises the Golgi dynamics (GOLD) domain (Anantharaman, 2002, Figure 3.2A). The GOLD domain is predicted to mediate protein-protein interactions and to form β-strands, exemplified by the crystal structure of SEC14L2 (Stocker, 2002). The p24 GOLD domain contains two highly conserved cysteines, one at each of its borders (Anantharaman, 2002).
The p24 proteins also carry a conserved short heptad repeat downstream of the
GOLD domain (Stamnes, 1995). This repeat is a postulated to form amphipathic coiled coils and to mediate oligomerization among p24 proteins (Ciufo, 2000).
The cytoplasmic tail of the p24s contains signal motifs that interact with coat proteins of COPI and COPII vesicles. A subset of these proteins contains the dilysine KKXX, KKXX-like or K(X)KXX motifs involved in COPI binding for
8 retrieval to the ER. The importance of the cytoplasmic tail motifs of p24s is further described in sections 1.2.3 and 1.2.4.
The p24 proteins are classified in subfamilies (α, β, γ, and δ) according to cluster tree based on their amino acid sequence (Dominguez, 1998). Although the p24s are similar to each other, their cytosolic tails display a higher degree of conservation whereas the luminal domains are more divergent. The roles of p24s in yeast and higher eukaryotes are described in sections from 1.2.2 to 1.2.7.
1.2.2 Yeast p24 proteins and their possible functions
There are eight members in the family of p24 proteins in S. cerevisiae. The first member, Emp24p, was discovered in yeast endosomes (Singer-Kruger, 1993), but was later determined that it predominantly localizes in the ER as the endosomal fractions were known to be contaminated by ER and Golgi fractions
(Schimmoller, 1995). Erv25p was also characterized to reside in the ER (Belden,
1996). The remaining six members, termed Erp1p to Erp6p, were identified and named (for Emp24p- and Erv25p-related proteins) by comparison of the first two members with the complete yeast genome sequence (Marzioch, 1999).
The yeast p24 proteins are not essential, as an eight-fold deletion strain grow like the wild type (Springer, 2000). The phenotypes shown by single,
Δemp24 (Schimmoller, 1995) and Δerv25 (Belden, 1996), four-fold (Δerp1, Δerp2,
Δemp24, and Δerv25, Marzioch, 1999), and eight-fold (Springer, 2000) deletion strains are the transport and maturation at reduced kinetics of Gas1p, an enzyme involved in cell wall maintenance, and Suc2p (invertase), a glycoprotein excreted into the periplasmic space; and Kar2p secretion into the medium. However, single
9 deletions of ERP3 to ERP6 show no particular phenotype (Marzioch, 1999). The
Kar2p secretion phenotype is caused by activation of the UPR (Belden, 2001a), thus, this pathway may cope for loss of p24 function. Furthermore, the existence of proteins that perform similar functions as the p24s might explain why the latter is not essential in yeast. Indeed, Yos9p (Yeast OS-9 homolog; Friedmann, 2002) and Ted1p (Trafficking of Emp24p/Erv25p-dependent cargo Disrupted; Haass,
2007) are also involved in the transport of Gasp1p. Thus, yeast cells have alternative pathways to achieve the same end.
Yeast p24s are involved in transport of secretory proteins as Emp24p and
Erv25p interact with Sec13p/Sec31p subcomplex of COPII vesicles probably with their FF motif in the cytosolic tail (Belden, 2001b). They serve as adaptors or receptors for Gas1p (Muniz, 2000) and probably invertase (Schimmoller, 1995).
In addition, Emp24p is implicated in concentrating GPI-anchored proteins at
ERES; and in Δemp24 cells, these proteins are retained in the ER (Castillon,
2008). Finally, the p24s become vital when GLO3, which encodes an ArfGAP involved in COPI vesicle formation (Lewis, 2004), is compromised due its genetic interaction with EMP24 and ERV25 (Aguilera-Romero, 2008). COPI formation is abolished in Δemp24 cells, which suggests that Emp24p is required for priming complexes for efficient polymerization of COPI coats because excess COPI proteins can cope for the loss of p24 function (Aguilera-Romero, 2008).
Several p24 genes are able to bypass the lethal phenotype of SEC13 deletion, which encodes a protein required for COPII vesicle biogenesis (Section
1.1.5). A mutation in EMP24 is able to restore the growth of Δsec13 cells (Elrod-
Erickson, 1996). Deletions of ERV25 and ERP1 are also capable to bypass SEC13
10 deletion (Marzioch, 1999). Thus, the p24s might be involved in regulation of vesicle coat assembly to cargo sorting (Elrod-Erickson, 1996).
The p24 proteins depend on each other for their stability and can associate into hetero-complexes (Marzioch, 1999). Emp24p and Erv25p interact with each other (Belden, 1996) and can associate with Erp1p and Erp2p (Marzioch, 1999).
However, Erp1p and Erp2 are not required for the incorporation of Emp24p or
Erv25p into COPII vesicles (Belden, 2001). Thus, Erp1p and Erp2 may just serve to link Emp24p/Erv25p complexes into dimers of dimers (Ciufo, 2000). The interactions of the other p24 members remain to be determined.
1.2.3 Mammalian p24 proteins
There are nine mammalian p24 proteins. The first one, gp25L (TMED9, hp241), was identified in microsomes of dog pancreas (Wada, 1991). Since then, several other members have been discovered and characterized: p23 (TMP21,
TMED10, hp241; Blum, 1996, Sohn, 1996), p24 (p24A, RNP24, TMED2, hp241; Stamnes, 1995), p25 (GMP25, ERS25, gp25L2, TMED4, hp242;
Dominguez, 1998), p26 (p24B, TMED3, hp244; Dominguez, 1998), p27 (gp27,
TMED7, hp243; Fullekrug, 1999), p28 (TMED5, hp242; Emery, 2000), Tp24
(TMED1, hp241; Gayle, 1996), and TMED6 (Clark, 2003).
Unlike yeast, p24 function in mammalian cells is essential as knockout of p23 is embryonic lethal in mice (Denzel, 2000). However, this lethal phenotype might be due to the mislocalization of essential proteins in the absence of p23
(Denzel, 2000). In multicellular organisms, the localization and expression of p24 proteins are cell-type specific. At steady state, in most cell types, most of them
11 (p23, p24, p26, p27, Tp24) are primarily found at the cis-Golgi (Dominguez,
1998; Blum, 1999; Fullekrug, 1999; Gommel, 1999), except for p25, which is more ER-localized (Dominguez, 1998; Emery, 2000). p24 and gp25L are mainly found at the ER of rat pancreatic acinar cells (Gommel, 1999), and dog prancreatic cells (Wada, 1991), respectively. Although they are predominantly localized in the early secretory pathway, where they cycle, the p24s can also be present at the PM of brain (Blum, 2008) and secretory granules of pancreatic cells
(Blum, 1996; Hosaka, 2007).
As in yeast, mammalian p24 proteins also have the ability to interact with each other. In fact, p24 coexpression is required for their ER exit (Dominguez,
1998; Fullekrug, 1999; Emery, 2000). Thus, the p24 proteins depend on each other for their proper localization. The interactions among p24s are specific as p27 interacts with p23, p24, and p25, but not p26 (Fullekrug, 1999; Lanoix, 2001).
Two interacting members, p23 and p24, are predominantly colocalized (Blum
1999; Gommel, 1999) and depend on each other for efficient transport of cargo proteins and members of other subfamilies cannot substitute in this process, but can increase the efficiency (Rojo, 1997; Emery, 2000).
The KK and FF motifs in the cytoplasmic tail of p24s are essential for their proper localization. Mutations from KK to SS of p23 (Emery, 2003) and p25
(Dominguez, 1998) not only mislocalize them to the PM, but the non-mutated p24s as well. Mutations from FF to AA of p23 and p25 retain them in the ER along with the wild type p24s (Dominguez, 1998). These facts strengthen the idea that p24s depend on each other for proper localization. Other abnormalities such
12 as mutations of the two C-terminal residues of p24 cytoplasmic tail from VV to
AA also lead to its mislocalization to the PM (Barr, 2001).
The mammalian p24s play important roles in the early secretory pathway.
Their involvement in the transport of cargo and the structure of organelles in the secretory pathway is described in section 1.2.4 and 1.2.5 respectively.
1.2.4 Involvement of mammalian p24 proteins in transport
The p24 proteins are present in COPII and COPI vesicles and cycle between the ER and Golgi in the early secretory pathway. There is limited characterization of the interactions of p24s with COPII proteins, but it was shown that p23, p24, p25, p26, and p27 interact with Sec23 (Dominguez, 1998).
In mammalian systems, at least in vitro, the p24 proteins are required for
COPI biogenesis as the latter would not occur in the absence of the former
(Bremser, 1999). First, a dimeric form of p23 recruits Arf1-GDP to the Golgi membrane and dissociates from Arf1 upon exchange from GDP to GTP by a GEF
(Gommel, 2001; Majoul, 2001). Coatomer (Section 1.1.6) is recruited to the membrane and interacts with Arf1-GTP via β-COP (Zhao, 1997) and dimers of p23 via γ-COP (Harter, 1999; Bethune, 2006a). Binding of ArfGAP1 is also likely mediated via γ-COP whereas binding of cargo proteins occurs via the α- and β’ subunits (Bethune, 2006a). Upon binding, the coatomer undergoes a conformational change, which drives the polymerization and formation of COPI vesicles (Reinhard, 1999). p23 is present in stoichiometric amounts with Arf1 and coatomer (Reinhard, 1999; Weidler 2000; Fligge, 2000). Moreover, COPI needs a bivalent interaction with p23 and Arf1 to be formed (Bremser, 1999).
13 GTP hydrolysis by Arf1 triggers the uncoating of the vesicles and should not happen before pinching off. Yet, GTP hydrolysis by Arf1 promotes selective segregation and concentration of cargo proteins into COPI (Lanoix, 1999). To explain this contradiction, it was proposed that GTP hydrolysis was required for
COPI priming, but once the coat starts polymerizing, the double arginine (RR) motif at the cytoplasmic tail of p24 inhibits ArfGAP1-mediated GTP hydrolysis to stabilize COPI until it is ready to pinch off (Lanoix, 2001).
The KKXX motif in the cytoplasmic tail of p24 proteins is responsible for the interaction with coatomer (Dominguez, 1998). This motif also exists in other proteins and is required for sorting and incorporation into COPI vesicles (Cosson,
1994). The binding is specific as p23 with its KKLIE and p25 with its true KKLV motif interact with coatomer with higher affinity than p24 with RRVV, which is homologous to KKXX (Sohn, 1996; Dominguez, 1998). Other p24s, such p27 are present in COPI vesicles as well, probably due to their interaction with proteins bearing a KKXX motif (Fullekrug, 1999; Lanoix, 1999). The FF motif N-terminal of KKXX serves to modulate retrograde transport (Nickel, 1997). Mutations of motifs from KK to SS and from FF to AA abolish binding to coatomer and impair retrograde transport (Nickel, 1997; Bremser, 1999).
Aside from COP vesicle components, the p24 proteins also interact with putative cargo proteins on which they act as receptors or adaptors. Recently, several potential cargos have been identified. For example, p24 interacts with
PAR-2, a member of the G-protein coupled protease-activated receptor (PAR) family, and is involved in the resensitization of this receptor on the cell surface
(Luo, 2007). Spastin, a member of the ATPases associated with various cellular
14 activities (AAA) family and involved in the autosomal dominant hereditary spastic paraplegia (AD-HSP; reviewed by Salinas, 2007), interacts with p25 (Reid,
2005). Atlastin, a dynamin-like large GTPase also involved in AD-HSP, interacts with p24 (Namekawa, 2007). SURF4, a human homolog of yeast Erv29p, interact with p23, p24, and p25 (Mitrovic, 2008). Finally, p24 proteins are required for efficient transport of GPI-anchored proteins from the ER to the cell surface
(Takida, 2008). Interestingly, ERS25 (p25) expression is induced under ER- specific stress, heat shock, and oxidative stress, which suggests it might interact with proteins involved in relieving these disturbances, pointing toward another direction for p24 function in the cell (Hwang, 2008).
1.2.5 Structural roles of mammalian p24 proteins
There is increasing evidence that p24 proteins play a structural role in the cell. Both p24 and p26 interact with GRASP55 and GRASP65, two Golgi structural proteins, through their cytoplasmic tail in an oligomeric fashion (Barr,
2001) and p27 is involved in early de novo assembly of Golgi (Stroud, 2003).
Knock down of p23 (Denzel, 2000; Rojo, 2000) and p25 (Mitrovic, 2008) with siRNA leads to a dispersed or fragmented Golgi apparatus. p25 may (by anti-p25 antibodies; Lavoie, 1999) or may not (by p25 siRNA knock down; Mitrovic,
2008) affect the generation of ERES structures. Moreover, p23 and p24 are involved in the formation of ER-to-Golgi tubular transport intermediates (TTIs) that are formed in the ERGIC (Simpson, 2006).
Overexpression of p24 proteins leads to the formation of large membranous perinuclear structures remisnescent of the ER and destruction of the
15 Golgi cisternal elements (Gommel, 1999; Rojo, 2000). The cells are still viable, but overexpressed p24s are relocalized to the ER even for non-overexpressed members (Gommel, 1999; Blum 1999). Perhaps, p24 overexpression saturates the
Golgi retention mechanism and causes delocalization to the ER by the retrograde pathway (Blum, 1999). However, this morphological change of ER and Golgi does not affect the transport or other proteins (Rojo, 2000; Mitrovic, 2008).
Thus, the p24 proteins have the intrinsic ability to control membrane composition and dynamics (Emery, 2003). The morphogenic ability of p24s contributes to the structural organization of organelles of the secretory pathway.
1.2.6 Alzheimer’s Disease and p24 proteins
TMP21 (p23) is involved in the pathogenesis of Alzheimer’s disease (AD;
Chen, 2006), which is characterized by the formation and accumulation of amyloid β-peptide (Aβ) plaques in brain cells. The Aβ peptides are generated by the plasma membrane-localized γ-secretase, which cleaves within the cell membrane a by-product from the activity of β-secretase on amyloid precursor protein (APP; reviewed by Steiner 2008). These peptides are toxic to the cell and form aggregates to generate plaques that deposit in brain cells. TMP21 negatively regulates the activity of the γ-secretase, and also interacts with it (Chen, 2006;
Vetrivel, 2007). In addition, TMP21 regulates the trafficking of APP (Vetrivel,
2007). However, TMP21 does not regulate the activity of neprilysin, a zinc- metalloprotease that degrades a number of secreted small peptides including Aβ
(Dolcini, 2008). Thus, there is some specificity in TMP21 function.
16 The components of the γ-secretase, which comprises presenilin-1 (PSEN1) or presenilin-2 (PSEN2), APH-1a or APH-1b, PEN-2, and nicastrin (NCSTN), are degraded through the ubiquitin-proteasome pathway; and TMP21, with a half-life of approximately three hours, is also degraded through this pathway (Liu, 2008).
Thus, the complex might be degraded as a whole (Liu, 2008).
TMP21 is widely distributed in different areas of the brain (Vetrivel,
2008). The high expression in brain cells leads to two different pools of TMP21: one that interacts with its p24 partners and one that associates with the γ-secretase.
TMP21 might be involved in trafficking of the complex to the cell surface and this event would be mediated by the luminal part of the protein (Blum, 2008).
Expression of TMP21 is decreased after birth, an event that may contribute to increased Aβ peptides production in the adult brain (Vetrivel, 2008).
Moreover, the cDNA clone (S31iii125) is similar to TMP21 and was identified within the AD3 locus on chromosome 14q24.3, which is associated with aggressive, early-onset AD (Sherrington, 1995; Liu, 2008). Therefore, this fact coupled with its function in regulating the activity of the γ-secretase suggests that
TMP21 may play an important role in AD pathogenesis. This discovery projects towards a novel road for investigation of the function of p24 proteins.
1.2.7 The p24 proteins of other organisms
There are nine p24 members in Drosophila melanogaster (Boltz, 2007): eclair (α), CG33105 (α), CG3564 (β), CG9308 (β), baiser (δ), CG1967 (γ),
CG9053 (γ), CG31787 (γ), and logjam (γ). As in mammalian cells, the p24 expression in fruit flies is tissue-specific and has specific developmental patterns
17 (Boltz, 2007). For example, logjam, the homolog of EMP24 in yeast, plays an essential role in female reproduction by modulating oviposition behavior as loss of function leads to sterility (Carney, 2003). Another example is that CG31787 is only expressed in males (Boltz, 2007). Loss of logjam function also causes stress response and upregulates the transcription of genes involved in relieving the stress, such as the NF-κB-regulated genes and those encoding proteins implicated in proteolysis, protein folding, and metabolism (Boltz, 2008).
There are six p24 genes in Caenorhabditis elegans: SEL-9 (β), F47G9.1
(δ), F57B10.5 (γ), K08E4.6 (γ), Y60A3A.9 (α), and T085D2.1 (α). SEL-9, a homolog of yeast EMP24, interacts genetically with LIN-12 and GLP-1, which are members of the LIN-12/NOTCH family of receptors involved in cell-cell interaction during development, and plays a role in their trafficking to the PM
(Greenwald, 1998). Reducing the expression of SEL-9 or F47G9.1 restores the trafficking to the cell membrane of a mutant GLP-1 that would otherwise accumulate within the ER (Wen, 1999). Thus, the p24 proteins play a role in quality control as well.
There are six p24 members in Xenopus laevis: p24α2, p24α3, p24β1, p24γ3, p24δ1, and p24δ2. Again, p24 expression is cell-type specific. The X.laevis p24s are involved in the trafficking of proopiomelanocortin (POMC) in active melanotrope cells, which are responsible for adaptation in black or white background (Kuiper, 2000; Kuiper, 2001; Rotter, 2002). Overexpression of some p24s mislocalizes a subset of endogenous p24s (Bouw, 2004; Strating, 2008). The p24 proteins also have a structural role, as they seem to form subcompartments with cargo proteins in the early secretory pathway (Strating, 2007).
18 1.3 Project proposal
Despite all the studies performed by different research groups over the years since the discovery of the first member in mammalian cells (Wada, 1991), the exact cellular functions of p24 proteins remain to be elucidated. Considering the fact that these small transmembrane proteins are remarkably conserved throughout evolution, they must play an important role. They are dispensable in yeast (Springer, 2000), but at least one member is essential in mice since a knockout is embryonic lethal (Denzel, 2000). Moreover, at least one member of the p24 family is involved in AD, characterized by the deposition of A plaques in brain cells, which suggests the importance of p24 proteins in proper cellular function (Chen, 2006).
Several roles have been predicted for the p24 proteins. They are involved in vesicle biogenesis (Goldberg, 2000; Lanoix 2001). They also serve as adaptors or receptors for cargo, especially GPI-anchored proteins, to be transported through the secretory pathway (Castillon, 2008; Takida 2008). There is increasing evidence that they also contribute to the structural organization of organelles of the early secretory pathway (Denzel, 2000; Emery, 2003). Most of the studies on these proteins were investigated on their cytoplasmic tail for their cellular localization and their function in vesicle formation, especially COPI in mammalian cells. Little has been done on their luminal domain, which is postulated to bind cargo proteins for incorporation into vesicles. On the other hand, the p24 proteins have the capacity to form complexes among them, but the pairwise interactions were not determined except in high-throughput screens
19 (Miller, 2005; Schuldiner, 2005; Gavin 2006, Krogan, 2006; Tarassov, 2008) in which only the full length p24s have been investigated.
Determination of individual p24 interactions among them and among their different domains would allow a better understanding of how these proteins associate and perhaps how they associate with their putative cargo. Since p24 deletion brings little insight in the role they play except for transport and maturation at reduced kinetics of some proteins (Gas1p and invertase) and ER chaperone (Kar2p) secretion, p24 overexpression might yield new clues about their cellular functions.
In this study, the interactions among p24 proteins for different domains
(full length, luminal domain, GOLD domain, and DOG (downstream of GOLD) sequence were determined using the MYTHS technique, a modified Y2H method
(Section 3.1, Figure 3.1A). Based on the results of the latter, one yeast p24 member was subjected to mutagenesis by PCR to attempt to define the interaction with its two partners. Furthermore, a yeast 3-hybrid experiment was performed to find out if overexpression of a p24 protein was able to bridge an interaction between two non-interacting p24s. Co-overexpression experiments in yeast were done to determine the effect on cells expressing large amounts of p24s and microscopy with fluorescent dyes was used to examine p24-coverexpressing cells exhibiting a phenotype. Finally, expression of p24 proteins for purification was performed in order to perform in vitro experiments for validation of MYTHS results. Here, I demonstrate that these experiments reveal new directions for investigation of p24 function in the cell.
20
Chapter 2
Material and Methods
21 2.1 Strains, cell lines, and media
The genotypes of Saccharomyces cerevisiae strains used in this study are listed in Table 2.1. Yeast and bacterial cells were grown in media listed in Table
2.2. For plates, 2% agar was added to the media. The concentration of antibiotics was 200g/mL and 50g/mL for yeast and bacterial media respectively. The initial insect cell Sf9 stock was obtained from Dr. B. Nagar (McGill University,
Montreal). The bacterial cells Escherichia.coli TOP10, BL21(DE3), CS, and
DH10Bac were rendered competent as described (Struhl, 2000).
2.2 Primers and plasmids
The primers used to amplify DNA sequences are listed in APPENDIX A.
The list of plasmids used in this study is provided in Table 2.3. A comprehensive list of the inserts in different plasmids is available in APPENDIX B. PCR reactions were done using the Expand High FidelityPLUS PCR System (Roche) and yeast BY4741 chromosomal DNA as template unless otherwise indicated.
2.3 Membrane yeast two-hybrid system (MYTHS)
2.3.1 Construction of yeast two-hybrid plasmids
Plasmids were made using the Gateway Cloning Technology with some modifications (Invitrogen). The DNA sequence was amplified with primers listed in Table A1 of APPENDIX A. The PCR product was purified using the QIAquick
PCR purification protocol (QIAGEN). A BP reaction was performed to insert the
PCR product into the entry vector. Essentially, 1µL of pDONR221 plasmid, 1µL of PCR product 2µL of Tris-EDTA (TE) pH 8.0, and 1µL of BP Clonase
22 Table 2.1 List of Strains Strain Genotype Source Y574 MATa ade2 trp1-901 leu2-3,112 his3-200 met gal4- G. Jansen gal80- Δire1::natR GAL(UAS)-HIS3, CYC1(2xUPRE)- LacZ::kanMX, CYC1(UPRE)- ADE2 Y575 MATα ade2 trp1-901 leu2-3,112 his3-200 met gal4- G. Jansen gal80- Δire1::natR GAL(UAS)-HIS3, CYC1(2xUPRE)- LacZ::loxP, CYC1(UPRE)- ADE2 EGY6 can1Δ::MFA1pr-HIS3-MFα1pr-LEU2 his3Δleu2Δ0 E. Garneau ura3Δ0 met15Δ0 lys2Δ erp4::natR-MX4 ETY1 MATa ade2 trp1-901 leu2-3,112 his3-200 met gal4- This study gal80- Dire1::natR GAL(UAS)-HIS3, CYC1(2xUPRE)- LacZ::kanMX, CYC1(UPRE)- ADE2 erp4::hygR-MX4 ETY2 MATα ade2 trp1-901 leu2-3,112 his3-200 met gal4- This study gal80- Dire1::natR GAL(UAS)-HIS3, CYC1(2xUPRE)- LacZ::loxP, CYC1(UPRE)- ADE2 erp4::kanR-MX4 W303- MATa leu2-3, 112, ura3-1 his3-11, 15 trp1-1 ade2-1 Thomas, 1A 1989 BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 Brachmann, 1998
Table 2.2 List of Growth Media Media Components YPADi 1% yeast extract, 2% peptone, 0.01% inositol, 2% dextrose, 0.01% adenine SC 0.67% yeast nitrogen base, 2% dextrose, 0.01% inositol, and 15mg/mL amino acids SD-X* 0.67% yeast nitrogen base, 2% dextrose, 0.01% inositol, and 15mg/mL amino acids with the appropriate dropout SD-X*-INO 0.67% yeast nitrogen base, 2% dextrose, and 15mg/mL amino acids with the appropriate dropout SD-X* 0.67% yeast nitrogen base, 2% raffinose, 0.01% inositol, and raffinose 15mg/mL amino acids with the appropriate dropout SD-X* 0.67% yeast nitrogen base, 4% galactose, 0.01% inositol, and galactose 15mg/mL amino acids with the appropriate dropout YPGal 1% yeast extract, 2% peptone, 0.01% inositol, 4% galactose supplemented with adenine LB 1% BactoTryptone, 1% sodium chloride, 0.5% BactoYeast Extract TB 1.2% Tryptone, 2.4% BactoYeast Extract, 0.4% glycerol, 17mM sodium phosphate monobasic, 72mM potassium phosphate dibasic *SD-X where X is the amino acid(s) to drop out
23 Table 2.3 List of plasmids Plasmids Description Reference pDONR221 KanR-CamR-ccdB, pUC, Entry vector Invitrogen pMYTHS3-SS LEU2-AmpR-CamR-NatR-ccdbB- Modified from Ire1p(K702R), Destination vector, 1 TMD, Jansen, 2005 Cen plasmid pMYTHS3+SS LEU2-AmpR-CamR-NatR-ccdB- Modified from Ire1p(K702R), Destination vector, 1 TMD, Jansen, 2005 IRE1 SS, Cen plasmid pMYTHS4-SS TRP1-AmpR-CamR-KanR-ccdB- Modified from Ire1p(D995A, K1058A), Destination vector, Jansen, 2005 1 TMD, Cen plasmid pMYTHS4+SS TRP1-AmpR-CamR-KanR-ccdB- Modified from Ire1p(D995A, K1058A), Destination vector, Jansen, 2005 1 TMD, IRE1 SS, Cen plasmid pMYTHS5-SS LEU2-AmpR-CamR-NatR-ccdB- Modified from Ire1p(K702R), Destination vector, 2 TMD, Jansen, 2005 Cen plasmid pMYTHS5+SS LEU2-AmpR-CamR-NatR-ccdB- Modified from Ire1p(K702R), Destination vector, 2 TMD, Jansen, 2005 IRE1 SS, Cen plasmid pMYTHS6-SS TRP1-AmpR-CamR-KanR-ccdB- Modified from Ire1p(D995A), Destination vector, 2 TMD, Jansen, 2005 Cen plasmid pMYTHS6+SS TRP1-AmpR- CamR-KanR-ccdB- Modified from Ire1p(D995A), Destination vector, 2 TMD, Jansen, 2005 IRE1 SS, Cen plasmid pHYPER3-SS LEU2-AmpR-HygR-ccdB-Ire1p(F980L) Modified from Destination vector, 1 TMD, Cen plasmid, Jansen, 2005 pHYPER3+SS LEU2-AmpR-HygR-ccdB-Ire1p(F980L) Modified from Destination vector, 1 TMD, IRE1 SS, Cen Jansen, 2005 pHYPER5-SS LEU2-AmpR-NatR-ccdB-Ire1p(F980L) Modified from Destination vector, 2 TMD, Cen plasmid Jansen, 2005 pHYPER5+SS LEU2-AmpR-NatR-ccdB-Ire1p(F980L) Modified from Destination vector, 2 TMD, IRE1 SS, Cen Jansen, 2005 p425 GALL-LEU2-AmpR, 2µ plasmid ATCC p426 GALL-URA3-AmpR, 2µ plasmid ATCC pADH426 ADH-URA3-AmpR, 2µ plasmid Mumberg, 1995 pFASTBac- AmpR, N-terminal SS for secretion into the Harvard Cpo-His6 medium and C-terminal 6xHis PlasmID pETDuet AmpR, 1st MCS N-terminal 6xHis, 2nd MCS Modified from modified C-terminal S-Tag Novagen pRSFDuet KanR, 1st MCS, cleavable N-terminal 8xHis Novagen with Tev protease, 2nd MCS C-terminal S-Tag
24 (Invitrogen) were mixed in an Eppendorf tube, vortexed briefly and incubated at
RT, overnight. The mixture was transformed (section 2.3.3) in E.coli TOP10 competent cells and plated onto LB medium containing 50µg/mL kanamycin
(Gibco). Plasmids of single colonies were isolated using the QIAprep spin miniprep protocol (QIAGEN) and digested with ApaI and EcoRV (New England
Biolabs). The digests were resolved by agarose gel electrophoresis and potential good clones were sequenced (IRIC, University of Montreal, Montreal).
The insert of the entry vector was then transferred into a destination vector by a LR reaction. The reaction mixture consisted of 1µL of destination vector,
1µL of entry vector containing the insert of interest, 2µL of TE pH 8.0, and 1µL of LR Clonase and was incubated at RT for 1 hour before transformation into
E.coli TOP10 competent cells (Section 2.3.3). Plasmids of single colonies were isolated and digested with restriction enzyme SalI. The good destination clones were transformed into yeast strains Y574 and Y575 (Section 2.3.4).
2.3.2 Verification of protein C-terminus orientation and expression level
The insert of pDONR221 was transferred into pHYPER3 and pHYPER5 destination vectors by LR reactions (Section 2.3.1) and transformed into Y574
(Section 2.3.4). Patches of transformants were replica plated onto SD-LAi and incubated at 30oC for 2 days. The latter step was repeated once more. The right orientation was assessed based on the growth of the cells. This system was also used to verify protein expression level. A reverse LR (same as a regular LR, insert transfer from destination to entry vector) may be required to insert a sequence from a destination vector to the entry vector before transfer into pHYPER vectors.
25 2.3.3 Bacterial transformation
For plasmid amplification, 1µL of plasmid DNA was added into 50µL of
E.coli TOP10 competent cells and the mixture was incubated on ice for 30 min and heat shocked at 42oC for 2 min. 1mL of LB was added and the tube was shaken at 37oC at 250 rpm for 1 hour. 50µL of the mixture was plated on LB with appropriate antibiotics. The plate was then incubated at 37oC overnight.
For BP or LR reactions, the total volume was added into 50µL of E.coli
TOP10 competent cells and the remainder of the procedure is described as above except that all the cells of the transformation mixture were plated.
2.3.4 Yeast transformation
Fresh cells were scraped from an YPADi plate and added to 100µL of
Transformation Mix (44% PEG, 0.1M lithium acetate buffered with TE to pH 8.0).
1µL of 5mg/mL sonicated salmon sperm carrier DNA (Stratagene) and 5µL of plasmid DNA were added to the cell suspension. The mixture was vortexed briefly and incubated at 30oC for a minimum of 2 hours before plating on selective media. The plates were incubated for 2 days at 30oC.
2.3.5 Yeast two-hybrid experiment (MYTHS)
A colony of each transformation of the same mating type was patched on selective medium and grown overnight to build the master plates. 5mL cultures in selective medium of each transformant of the other mating type were grown overnight. The cells of the cultures were plated onto YPADi plates. Master plates were replica plated onto the YPADi plates, which were incubated at 30oC overnight. Diploids were selected by replica plating onto SD-LT dextrose plates
26 and incubated for 2 days at 30oC. The diploids were replica plated onto SD-LTAi dextrose and incubated for 2 days at 30oC. The latter step was repeated once more.
2.4 Random mutagenesis by PCR
2.4.1 PCR experiment to generate random mutations
The PCR reaction mixture consisted of 5µL of 2.5mM dNTPs mix
(Stratagene), 5µL of 10X PCR Buffer, 1.5µL of 50mM magnesium chloride, 10µL of 5mM manganese chloride, 1µL of template from entry vector, 0.5µL of each primer (Table A2 of APPENDIX A), 0.5µL of Taq Polymerase (Invitrogen), adjusted to a volume of 50µL with sterile water. The PCR cycle was performed as follows: 2 min of denaturation at 95oC, followed by 35 cycles of three amplification steps, which started with 30s at 95oC, followed by 30s at 54oC, and
1 min at 68oC, and finally a 5-min incubation step at 68oC.
2.4.2 In vivo recombination
Y574 or Y575 cells were transformed as in section 2.3.4 except that 1µL of overnight SalI-digested destination vector and 15µL of PCR product (Section
2.4.1) were used and that the transformation mixture was incubated overnight at
30oC. Moreover, the plating was performed as follows: 400µL of selective medium was added to the transformation mixture and 50µL was plated on 10 separate selective medium plates and incubated for 3 days at 30oC.
2.4.3 Mutants screening
MYTHS experiments were performed as described in section 2.3.5 except that the master plates were the plates with transformants from section 2.4.2. The
27 colonies were mated with cells containing the plasmid of one of the two interactors and the empty vector. After the second replicate on SD–LTAi dextrose medium, the colonies that grew when mated with cells of one partner, but not the other, were considered potential mutants. These colonies were picked from their corresponding haploid cells and the MYTHS experiment was repeated. Plasmids of potential mutants were then extracted (Section 2.4.4) and transformed into bacteria for amplification (Section 2.3.3). Plasmids were extracted from bacteria following the QIAprep miniprep protocol (QIAGEN) and retransformed into yeast
Y574 or Y575 (Section 2.3.4). The MYTHS procedure was repeated again and the plasmids with potential good mutants were sequenced to locate the mutations.
2.4.4 Plasmid DNA extraction from yeast
Fresh yeast cells containing the plasmid of interest were scraped from an agar plate and added to 250µL of SEZ solution (1mg/mL zymolase (100,000U/g,
MP Biomedicals) in 1.2M sorbitol/50mM EDTA), vortexed, and incubated at
37oC for 1 hour to digest the cell wall. The standard QIAprep miniprep protocol
(QIAGEN) was used to perform the extraction except that the plasmid DNA was eluted with 22L of warm elution buffer (10mM Tris-HCl pH 8.5).
2.5 Yeast three-hybrid
2.5.1 Knockout by gene replacement
KanMX and HygMX cassettes were amplified by PCR (Primers in Table
A3 of APPENDIX A) using pMYTHS4-SS and pHYPER3-SS as template respectively. The PCR products were transformed separately into EGY6 cells
28 (Garneau, 2004) using the high efficiency yeast transformation protocol (Section
2.5.2). The purpose of this step was to replace the clonNAT cassette by that of
KanMX or HygMX. Overnight cell patches from transformants were replica plated onto YPADi containing NAT and incubated overnight at 30oC. The chromosomal DNA of cells that did not grow on YPADi containing NAT was extracted (Section 2.5.3). A PCR was performed on the chromosomal DNA to verify the cassette replacement by agarose gel electrophoresis and to amplify the cassette itself (Primers in Table A4 of APPENDIX A). The PCR product of
HygMX and KanMX cassettes was transformed into Y574 and Y575 respectively
(Section 2.5.2). The chromosomal DNA of transformants was extracted (Section
2.5.3). A PCR was performed to amplify the ORF of ERP4 (Primers in Table A4 of APPENDIX A). The PCR products were digested by restriction enzymes: BglII and HincII for HygMX cassette, BglII and HindIII for KanMX cassette, and SalI and EcoRI for ERP4. The erp4 cells (termed ETY1 from Y574 and ETY2 from
Y575) were used for Y3H experiments (Section 2.5.4).
2.5.2 High efficiency yeast transformation
A volume of 125µL of YPADi overnight cell culture was inoculated to
5mL fresh YPADi and grown at 30oC with shaking for 5 hours. Cells were washed with 5mL of sterile water followed by 1mL of lithium acetate buffer
(0.1M lithium acetate, TE buffer at pH 8.0). Cells were resuspended in 100µL of
Transformation Solution (Section 2.3.3) followed by addition of 15µL of PCR product and 1µL of salmon carrier DNA. The transformation mix was incubated
29 overnight at 30oC and recovered in 1mL YPADi for 3 hours, shaking at 30oC before plating onto YPADi plates containing appropriate antibiotics.
2.5.3 Yeast chromosomal DNA extraction
Fresh cells were scraped from a plate and resuspended in 600µL SEZ
(Section 2.4.4). The mixture was vortexed briefly and incubated at 37oC for 1h30 followed by a centrifugation step of 10 min at 300g. The supernatant was discarded and the remainder of the procedure was performed following the protocol provided by the manufacturer (DNeasy Tissue Kit, QIAGEN).
2.5.4 Yeast three-hybrid experiment
The sequence of ERP4 was PCR amplified (Primers in Table A5 of
APPENDIX A) and inserted into the pADH426 vector using the BamHI and XhoI sites. Y574 and ETY1 cells were transformed with pMYTHS plasmids; Y575 and
ETY2 with pADH426 plasmid (Section 2.3.4). The Y3H experiment was done as in section 2.3.5 except that the selective media were SD-LTU and SD-LTUAi.
2.5.5 Plasmid loss
Fresh cells from selective medium were inoculated in 5mL of SC and incubated at 30oC with shaking for 4 days with a 5µL transfer of the overnight culture to 5mL of fresh SC every day. 500 cells were plated onto SC and incubated for 2 days at 30oC. The plates were replica plated on SD-LEU, SD-TRP, and SD-URA and incubated overnight at 30oC followed by replica plating each of those plates on SD-LT, SD-LU, and SD-TU, which were also incubated at 30oC overnight. A colony from each plate were patched onto a SC plate and incubated
30 overnight at 30oC. The SC plate was replica plated onto SD-ADE and incubated at
30oC overnight. This step is repeated three more times. The SC plate was also replica plated onto different selective media to verify the presence of plasmids.
2.6 Co-overexpression in yeast
2.6.1 Cloning and transformation into yeast
Sequences were PCR amplified using yeast BY4741 chromosomal DNA or human brain cDNA as template (primers in Table A5 of APPENDIX A) and inserted into p425 and p426 vectors using BamHI and XhoI sites. Plasmids were transformed into W303-1A cells (Section 2.3.3). Patches of colonies were replica plated onto YPGal and incubated overnight at 30oC to select for pink colored cells.
2.6.2 Serial dilution screen
Cells were grown in 200µL of selective medium containing 2% raffinose overnight at 30oC. They were serially diluted 7 times with a factor of 5 with selective medium containing 4% galactose for a volume of 100L per dilution per sample in 96-well plates. The plates were incubated for 120 hours at 30oC with scanning and measuring OD600 every 24 hours. The serial dilutions were done with the BioMek FX Laboratory Automation Workstation (Beckman Coulter).
2.6.3 Growth curves
Overnight cultures grown in selective medium containing 2% raffinose were diluted to an OD600 of 0.05 in 5mL of selective medium containing 4% galactose. OD600 was monitored every few hours for 96 hours.
31 2.6.4 Recovery in dextrose
Aliquots of 5000 cells from overnight cultures grown in selective medium with 2% raffinose were inoculated into 100µL of selective medium with 4% galactose and incubated at 30oC. After every 24 hours, 5000 cells were plated onto selective medium with 2% dextrose. Plates were incubated for 2 days at 30oC.
2.6.5 Cell size assessment by microscopy
Cells were grown overnight in 5mL selective medium containing 2% raffinose. 0.5mL of the overnight culture was added to 5mL of selective medium containing 4% galactose. Cells were incubated at 30oC with shaking for 2 days.
The cell size was assessed by brightfield microscopy using a Plan-NeoFluar 100X oil immersion lens of an AxioSkop machine (Carl Zeiss Canada).
2.6.6 Staining with fluorescent dyes
Cells were grown as described in section 2.6.5 and 250µL of each culture was subjected to separate staining. For Hoechst 33258 (Sigma) staining, cells were washed twice with sterile water and fixed with 1mL of 70% ethanol for 10 min. The fixed cells were washed twice with water following by three other washings with PBS. Cells were resuspended with 1mL PBS and 1µL of 500ng/mL
Hoechst 33258 dye. Cells were washed again as in after fixation step and resuspended with 100µL PBS. Fluorescence was monitored under the DAPI filter.
For Calcofluor White (Sigma) staining, cells were washed twice with sterile water and were resuspended with 10µL of 20mg/mL Calcofluor White dye.
The resuspension was incubated at RT for 10 min with agitation. Cells were
32 washed twice with sterile water and resuspended in 100µL water. Fluorescence was monitored under the DAPI filter.
The Rhodamine 123 (Sigma) staining was performed as described (Egner,
1998). FUN-1 (Invitrogen) staining was done essentially by following the protocol from the manufacturer. Briefly, cells were stained for 30 min at 30oC followed by removal of the dye from the media, and cells were resuspended with
100µL staining buffer without the dye.
2.7 Expression in insect Sf9 cells
2.7.1 Cloning into plasmid and conversion into bacmid
The Bac-to-Bac® Baculovirus Expression Systems (Invitrogen) was used to express proteins in Sf9 insect cells. Sequences were PCR amplified (primers in
Table A6 of APPENDIX A, entry vectors as template) and inserted into the pFASTBac11-Cpo-His6 plasmid using the RsrII restriction site. The plasmids were transformed into E.coli DH10Bac cells essentially as described by the manufacturer (Invitrogen) for conversion into bacmids. Single colony was inoculated into 200mL LB containing antibiotics and incubated overnight at 37oC.
The bacmid was extracted by following the QIAfilter Plasmid Purification protocol (QIAGEN). The DNA pellet after the final wash was dissolved with 10 mM Tris-HCl pH 8.5 to obtain a concentration of 1µg/µL.
2.7.2 Bacmid transfection and large-scale infection of Sf9 insect cells
Transfections were performed using CellFECTIN® essentially according to the indications of the manufacturer (Invitrogen). The final medium, termed P1 and
33 which contains the baculovirus, was collected and cleared of cells by centrifugation at 2200 rpm, RT. The cells were used to verify the expression of the protein of interest by SDS-PAGE and Western blot (Section 2.10).
Amplification of the baculovirus stock was done by infecting 2 x107 cells seeded in a T75 flask with 1mL of the P1 stock to yield the P2. The large-scale infection of Sf9 cells was essentially carried out as described (Tessier, 1999) except that the cells were incubated at RT.
2.7.3 Purification of protein secreted into the medium by infected Sf9 cells
The medium was separated from the cells by centrifugation and adjusted to 40mM Tris-HCl pH 7.5, 500mM sodium chloride using a Tangential Flow
Filtration (TFF) capsule with 10 kDa molecular weight cut-off (Pall Corporation).
The medium was then concentrated down to 10mL using the same TFF system following the procedure from the manufacturer. The protein was purified with Ni-
NTA beads as described in section 2.9.1 except for the content of the Wash Buffer
(40mM Tris-HCl pH 7.5, 500mM sodium chloride) and Elution Buffer (40mM
Tris-HCl pH 7.5, 500mM sodium chloride, 250mM imidazole).
2.8 Coexpression in bacterial cells
2.8.1 Cloning with dual expression vectors
Sequences were PCR amplified (Primers in Table A7 of APPENDIX A, entry vectors as templates) and inserted into plasmids pRSFDuet and pETDuet- modified. Plasmids were transformed into BL21(DE3) E.coli cells ( Section 2.3.3 as for plasmid amplification) for large-scale expression and purification.
34 2.8.2 Large-scale coexpression
Single colony was inoculated in 150mL of LB with antibiotics and incubated at 30oC overnight shaking at 250 rpm. 25mL of the overnight culture was added to each liter of TB with antibiotics for a total of 4L. The cultures were grown to an OD600 of 0.4-0.6 and were induced by addition of 0.1mM IPTG. The cultures were incubated overnight at 23oC shaking at 250 rpm. Cells were harvested by centrifugation at 6000 rpm for 10 min. Pellets were kept at -80oC.
2.9 Protein Purification
2.9.1 Ni-NTA purification of His6-tagged proteins
Pellets were resuspended in Lysis Buffer (50mM Tris-HCl pH 7.5, 50mM sodium chloride, 1mM β-mercaptoethanol (Sigma-Alrich), 0.1% Triton-X-100
(Fisher Scientific), 100µg/mL lysozyme (Fluka), protease inhibitors cocktail
(Roche)) and incubated for 1 hour on ice. The resuspension was then sonicated
(VibraCell, Sonics & Materials) for 30s with pulse for 7 cycles with cooling of probe between each cycle. The lysate was centrifuged at 15,000 rpm at 4oC for 30 min. The supernatant was transferred to a fresh tube and further centrifuged at
18,000 rpm at 4oC for 30 min. The cleared supernatant was filtered by gravity with Whatman P8 filter paper. The filtrate was mixed with equilibrated Ni-NTA beads (QIAGEN, 2mL of 50% slurry), at 4oC, rocking for 1 hour. The beads were allowed to settle. The filtrate was let flow through the column. The beads were washed twice with 20 CV of Washing Buffer (50mM Tris-HCl pH 7.5, 50mM sodium chloride). The protein was eluted with 5 x 1mL of Elution Buffer (50mM
35 Tris-HCl pH 7.5, 50mM sodium chloride, and 250mM imidazole). Samples for
SDS-PAGE were prepared for each step of the purification process (Section 2.10)
2.9.2 Dialysis of Ni-NTA purified proteins
Protein from pooled elution fractions was dialyzed overnight at 4oC in 2L of dialysis buffer (20mM potassium phosphate pH 7.5, 20mM sodium chloride).
2.9.3 Mono Q chromatography
Dialyzed protein was passed through a 5/50 GL mono QTM column (GE
Healthcare) using the ÄKTA Purifier apparatus (GE Healthcare). The protein was eluted using a gradient up to 600mM of sodium chloride for a CV length of 15 with 0.5mL per fraction. SDS-PAGE was performed to locate the protein.
2.9.4 Gel filtration
The protein solution was passed through a HiLoadTM 16/60 SuperdexTM
200 prep grade gel filtration column (GE Healthcare). An isocratic elution of 0.25
CV was done before fractionation (3mL per fraction) for 0.75 CV. SDS-PAGE was done to locate the protein.
2.10 SDS-PAGE and Western blotting
Samples were resolved on 15% polyacrylamide gels by SDS-PAGE in running buffer (25mM Tris base, 192mM glycine, 0.1% SDS) and were transferred onto a nitrocellulose membrane (Transfer-Blot, Biorad) in transfer buffer (25mM Tris base, 192mM glycine, and 20% methanol) using a Biorad
Mini-Gel apparatus. The membrane was blocked in 5% skim milk buffered with
TBS-T (10mM Tris-HCl pH 7.4, 10mM sodium chloride, 0.1% Tween-20) for 1
36 hour at RT. The membrane was incubated with shaking in rabbit polyclonal anti-
His probe (Santa Cruz Biotechnology) diluted 1:1000 in TBS-T with 5% skim milk for 1 hour at RT. Unbound antibodies were removed by 4x5 min washes with TBS-T containing 5% skim milk. The membrane was incubated for 1 hour in horseradish peroxidase conjugated goat anti-rabbit secondary antibody (Santa
Cruz Biotechnology) diluted 1:10,000 in TBS-T with 5% skim milk followed by
4x5 min washes. The signal was detected with SuperSignal West Pico Substrate
(Pierce Biotechnology) followed by exposure to photographic film (Kodak).
2.11 Pull-down assays
2.11.1 Coupling of protein of interest to beads
If the protein solution contains an amino group in buffer, a 4x4mL buffer exchange to HEPES/Acetate Buffer (25mM HEPES pH 7.0, 115mM potassium acetate, 0.75mM calcium chloride) was performed by centrifugation at 4000 rpm at 4oC for 30 min per exchange. Aliquots of 100µL of NHS-activated
SepharoseTM 4 Fast Flow beads (Amersham Biosciences) were prepared in 1.5mL microfuge tubes. Beads were washed four times with 500µL ice-cold 1mM hydrochloric acid (centrifuge at 3000 rpm for 30s at 4oC between each wash).
After removal of final wash, 500µL of protein solution (1mg/mL) was added to beads and the mixture was incubated at 4oC, rocking, for 2 hours. The mixture was further incubated at RT, rocking for 30 min. The beads were incubated with
0.5mL Blocking Solution (0.5M ethanolamine pH 8.3, 0.5M sodium chloride) for
30 min at RT and washed with 4x1mL Hepes/Acetate buffer.
37 2.11.2 Rat smooth ER preparation
Rat smooth ER (Ali Fazel, McGill University, Montreal) was diluted 10- fold with HEPES/Acetate buffer (Section 2.11.1) containing 0.1% Triton-X-100 and solubilized for 20 min at 4oC, rocking. The solubilized ER was centrifuged at
10,000g for 10 min at 4oC. The supernatant was used for pull-down.
2.11.3 Pull-down experiment
An aliquot of 100µL of solubilized rat smooth ER (Section 2.11.2) was added to 10µL of protein-coupled beads (Section 2.11.1). The mixture was incubated at 4oC for 2 hours with rocking. The beads were washed with 900µL of
HEPES/Acetate buffer, centrifuged at 5000 rpm for 30s and the supernatant was aspirated. The wash was repeated with 1.2mL of HEPES/Acetate buffer. 50µL of
2X SDS sample buffer (150mM Tris-HCl pH 6.8, 1.2% SDS, 30% glycerol, 15% v/v β-mercaptoethanol, bromophenol blue) was added to the beads and the tubes were shaken at 37oC for 5 min. The beads were centrifuged at 13,000 rpm for 5 min. The supernatant was transferred to a fresh tube and was analyzed by SDS-
PAGE, silver staining (Section 2.11.4), and MS (McGill University, Montreal).
2.11.4 Silver staining
The revolving polyacrylamide gel was fixed with 100mL destain (50% methanol, 10% acetic acid) for 2 hours and washed with deionized, distilled water for 1 hour. It was then incubated with 100mL of 0.02% sodium thiosulfate for 1 min and rinsed with water twice for 1 min. The gel was incubated with 100mL of
0.1% silver nitrate containing 0.04% formalin for 20 min and rinsed twice with water for 1 min. The gel was developed in 100mL of 2% sodium carbonate
38 containing 0.04% formalin until desired staining intensity was obtained. The gel was incubated for 2 min with 100mL of 5% acetic acid, washed with water for 5 min and stored in gel preserving solution (25% methanol, 2% glycerol).
2.12 Crystallization screen
The suites of Classics I, Classics II, Sparse Matrix II, and PEGS (all from
QIAGEN) were used to set up an initial crystallization screen by the Phoenix
Liquid Handling System robot (Art Robbins Instruments). Briefly, 100µL of screening solution was added to a reservoir of a condition. Two wells of each condition were inoculated with a drop (0.1µL) of the protein at either 15mg/ml or
30mg/ml. The plates were incubated in a stable room and formation of crystals was monitored under a light microscope.
2.13 NMR experiment
The NMR experiment was performed at Québec/Eastern Canada High
Field NMR Facility (McGill University, Montreal). The NMR spectrum was recorded on Varian INOVA 500 MHz spectrometer for 1H at 37oC. The protein concentration was 10mg/mL in buffer (20mM sodium phosphate pH 7.50 and
20mM sodium chloride) containing deuterated water.
2.14 Circular dichroism
CD experiments were performed with a Jasco-810 spectrometer
(Concordia University, Montreal). Spectra were recorded in triplicate at 50nm/s from 260 to 180 nm at 37oC.
39
Chapter 3
Results
40 3.1 Interactions of yeast p24s determined by MYTHS
Although it is established that p24 proteins form hetero-complexes in both yeast and mammalian cells (Marzioch, 1999; Fullekrug, 1999), the pairwise p24 interactions between members of the family were not determined except in large- scale screens, in which only the full length p24s were examined (Miller, 2005;
Schuldiner, 2005; Krogan, 2006; Tarassov, 2008). In order to determine the interactions between the eight members of yeast p24s (Erp1p-Erp6p, Emp24p, and Erv25p), a modified Y2H technique, the ER membrane yeast two-hybrid system (MYTHS; Pollock, 2004) was used. The MYTHS is based on the principle of the UPR (Section 1.1.1) and described in Figure 3.1A. In this study, MYTHS experiments were performed independently in triplicate for both the bait and the prey (also defined as both orientations) for each of the p24s. Only the bait-prey combinations that exhibited growth in all six replicates were considered to be interacting. An example of a MYTHS experiment is provided in Figure 3.1B. As a control, each p24 fusion protein was also tested with pMYTHS empty vector to detect any background growth. The signal transducing MEK kinase Ste11p and the adaptor protein Step50p were used as positive controls and to compare the growth and strength of interaction since they interact with each other (Ramezani,
1998). Moreover, Ste11p and Ste50p were used to test all p24 fusion proteins for both orientations in order to define the threshold of the background, as Ste50p is a protein that exhibits background growth in MYTHS experiments (laboratory unpublished observations). If there was growth for cells with Ste50p and a p24 fusion protein and that this growth was comparable to the growth of cells with the same p24 and another p24, then these two p24s were considered to be non-
41 interacting. It should be noted expression using pMYTHS plasmids is under control of the native yeast IRE1 promoter to prevent high expression of proteins in order to avoid occurrence of non-specific interactions (Pollock, 2004). The pLJ88-IRE1 vector was used as a positive control for the reporter.
Different sequences of p24 genes were inserted into pMYTHS plasmids
(Figure 3.2A). Since t he full length p24s have a transmembrane domain (TMD), a signal sequence (SS) for insertion into ER membrane, and a C-terminus in the cytoplasm, their sequences were inserted into pMYTHS5-SS and pMYTHS6-SS to bring the kinase/RNAse domains of Ire1p to the cytosol (Figure 3.1C). The luminal and GOLD domains were in pMYTHS3-SS and pMYTHS4-SS since they do not bear a TMD and their C-terminus was inside the ER lumen. The DOG sequences were in pMYTHS3+SS and pMYTHS4+SS because they do not have their native SS.
The interactions among yeast p24s were assessed in the following combinations: Full length with full length, luminal with luminal domains, GOLD with GOLD domains, and DOG with DOG sequences. In general, there were sixteen interactions among the luminal domains, including five self-interactions
(Figure 3.2B). Eleven interactions were detected among the GOLD domains, two of which were self-interactions (Figure 3.2C-D). Finally, there were only two interactions among the DOG sequences, including one self-interacting (Figure
3.3A). However, the interactions of the full length yeast p24s could not be fully determined as there was an expression problem with ERV25 (Figure 3.3B-C).
Excluding this issue, there were ten interactions, including three self-interacting.
42 Figure 3.1. The principle of the membrane yeast two-hybrid system
(MYTHS) and the hyperactive RNAse system
A. The MYTHS principle is based on the UPR described in section 1.1.1 and is
more detailed elsewhere (Pollock, 2004). Briefly, the ER luminal domains of
Ire1p were replaced with bait and prey proteins of interest. If the two proteins
interact, the cytosolic kinase domains of Ire1p to which the proteins of interest
are fused to will dimerize and activate transphosphorylation. The RNAse
domain will cleave HAC1 mRNA, which triggers its translation. Hac1p will
activate the transcription of the reporter, the ADE2 gene. Only cells with
interacting bait and prey will grow on medium lacking adenine. A mutation
was introduced in the RNase domain for the bait and in the kinase domain of
the prey to prevent self-interaction of the bait or the prey.
B. An example of MYTHS experiment of yeast p24s. Only the combinations that
showed growth in both orientations (bait and prey and vice-versa) and more
growth compared to testing with the controls (EV, Ste11p, and Ste50p) for
background were considered to be positive. In this example, both Erp1G and
Erp6G interact with Erp4G.
C. The hyperactive RNAse system. A mutation (F980L) in the RNAse of Ire1p
renders it constitutively active, which can be used to verify the C-terminus
orientation of the protein in order to determine the pMYTHS plasmid to use
and to verify the level of protein expression. II and III have no signal because
the RNAse domain is in the ER lumen. I and IV are able to activate the
reporter since the RNAse domain is in the cytosol.
43 AAA Hac1p A HAC1u B UPRE ADE2 pMYTHS4-SS
Erp1G Erp4G Erp6G EV RNAse Erp1G P P P P Kinase Cytosol Erp4G
bait prey bait prey bait prey ER Lumen Erp6G pMYTHS3-SS pMYTHS3-SS EV
Hac1p Ste11p Ire1p AAA Ste50p C HAC1u UPRE ADE2 I II III IV pMYTHS4-SS RNAse * *
EV Erp1G Erp4G Kinase Erp6G Ste50p Cytosol pMYTHS3-SS Ste11p pMYTHS4-SS ER Lumen Ste50p
EV
* * Erp1G Erp4G Erp6G Ste11p
= protein of interest = TMD of protein of interest = TMD of plasmid pMYTHS3-ss
Figure 3.2. Interaction maps of the luminal and GOLD domains of yeast p24 proteins
MYTHS experiments were performed for the luminal and GOLD domains of the eight members of yeast p24 proteins. The p24 members are color-coded according to the family in which they belong to. L = luminal, G = GOLD.
A. Domain structure of a p24 protein. Interactions of p24s were tested among
their luminal domain, GOLD domain, DOG sequence, and full length.
B. Interaction map of the luminal domains of yeast p24 proteins
C. Interaction map of the GOLD domains of yeast p24 proteins
D. An alternate view of the interaction map of the GOLD domain of yeast p24
proteins
45 A B Erp1L Erp2L Erv25L Erp4L Erp5L
Emp24L N SS GOLD DOG TM C Erp6L Luminal Erp3L α β C D Erp1G Erp1G γ Erp2G Erv25G δ Erp5G Erp2G Erp4G Erp4G Erp3G Emp24G Erp6G Erp5G Erp6G
Erv25G Erp3G Emp24G Overall, the interactions detected among the GOLD domains fairly resembled to those of the luminal domains (compare Figure 3.2B-C). The differences were that the luminal domains of Erp1p, Erp3p, and Erp6p self- interacted, whereas their GOLD respective domains did not. Other interactions that were present in the luminal domain, but not the GOLD domain, were those of
Erp1p and Erp5p, Erp1p and Erv25p, and Erp6p and Erv25p. The GOLD domain interaction between Erp2p and Erp6p was not seen in their luminal domain.
It is interesting to note that in the GOLD domain map, except for the self- interactions of Erp5G and Erv25G, members of the same families did not interact with each other. Moreover, Erp1G and Erp6G interacted with all members of the
-family and Erp3G interacted with all members of the α-family.
Since there were differences in the interactions among the GOLD and among the luminal domains, I decided to test the interactions between these domains (Table 3.1) and both of them with DOG sequences (Table 3.2). The results proved to be interesting. For example, Erv25G interacted with Erp1L, but
Erp1G did not interact with Erv25L. This observation could suggest that Erp1p requires both its GOLD domain and DOG sequence to be able to interact with the
GOLD domain Erv25p. However, upon performing MYTHS experiments with the
DOG sequences, I found out that it was not the case as Erp1D interacted with both
Erv25G and Erv25L; and Erv25D did not interact with Erp1G or Erp1L. This finding suggests that the DOG sequence of Erp1p is responsible for the interaction with the GOLD domain of Erv25p. All the others interactions included the GOLD
47 domain whether the DOG sequence contributed or not, but interestingly a lot of them only depended on the GOLD domain (Table 3.3).
To conclude, it can be safely speculated that the interactions among p24 proteins occur through their GOLD domain with some contribution from the DOG sequence (Figure 3.3D). Moreover, there is some specificity in the interactions as not every p24 protein interacts with all other p24s.
Table 3.1 Interactions between GOLD and luminal domains of yeast p24 proteins GOLD domain (G) – bait Luminal domain (L) – prey Erp1G Erp1L, Erp2L, Erp3L, Erp4L Erp2G Erp1L, Erp6L Erp3G Erp1L, Erp5L, Erp6L Erp4G Erp1L, Erp6L Erp5G Erp1L, Erp3L, Erp5L, Erv25L Erp6G Erp3L, Erp4L, Erp6L Emp24G Erv25L Erv25G Erp1L, Erp5L, Erp6L, Emp24L, Erv25L
Table 3.2 Interactions of DOG sequences with GOLD and Luminal domains of yeast p24 proteins DOG domain (D) – GOLD domain (G) – Luminal domain (L) – bait prey prey Erp1D Erv25G Erp3L, Erv25L Erp2D None None Erp3D Erp5G Erp1L, Erp3L, Erp5L, Erp6L, Erv25L Erp4D None None Erp5D None None Erp6D None Erp3L Emp24D Erv25G Erv25L Erv25D None Erp6L
Table 3.3 Interactions of yeast p24 proteins that only depend on the GOLD domain Erp1G-Erp2G Erp4G-Erp6G Erp1G-Erp4G Erp5G-Erp5G Erp2G-Erp6G Erp5G-Erv25G Erp3G-Erp6G Erv25G-Erv25G
48
Figure 3.3. Interaction maps of the DOG sequences and full length of yeast p24 proteins
MYTHS experiments were performed on the DOG sequences and the full length of the eight members of yeast p24 proteins. The p24 members are color-coded according to the family in which they belong to. D = DOG.
A. Interaction map of the DOG sequences of yeast p24 proteins.
B. Interaction map of the full length yeast p24s. Erv25p was not expressed as
verified by the hyperactive RNAse system.
C. An alternate view of the interaction map of the full length yeast p24s. Erv25p
was not expressed as verified by the hyperactive RNAse system.
D. A model of interaction between the p24 proteins. The interactions between
yeast p24 proteins occur mostly through their GOLD domain with some
contribution from the DOG sequence.
49 A B Erp2D Erp5D Erp1p Erv25D Erp2p α Erv25p Erp4D Erp1D β Erp3D Erp4p Erp5p Emp24p Erp6D Emp24D γ Erp6p δ
ERp3p C Erp1p D
Erp3p Cytosol
Erp2p ER DOG DOG
Erp5p Erp6p GOLD GOLD
Erp4p Erv25p p24 p24 Emp24p 3.2 Interactions of human p24s determined by MYTHS
The p24 family of proteins is conserved throughout evolution. It was of interest to find out if the human p24 proteins would interact in the same manner as their yeast counterparts. To find out, MYTHS experiments were performed and interactions were evaluated as for the yeast p24s (Section 3.1). Only the luminal domains of human p24s were considered at first. Among the nine members of human p24s, there were only one self-interaction (p24A) and one interaction between TMP21 and p24A (Figure 3.4), which are homologs of yeast Erv25p and
Emp24p respectively (Dominguez, 1998). I then used MYTHS to test the interactions between the GOLD domains of TMP21 and p24A and the same results as for the luminal domains were observed (Figure 3.4). Thus, the interactions among human p24 proteins also occur through the GOLD domain.
3.3 Components of the -secretase
TMP21 is involved in AD by negatively regulating the activity of the γ- secretase and immunoprecipitation experiments have shown that it is part of the complex, which consists of PSEN1 or PSEN2, NCSTN, PEN-2, and APH-1
(Chen, 2006; Vetrivel, 2007). To determine the interactions between those components and TMP21, MYTHS experiments were performed. The sequences of the γ-secretase components were inserted into appropriate pMYTHS plasmids
(Table B2 of APPENDIX B) depending on their topology described in the literature. Only that of PSEN2 was not characterized and the TMHMM server
(http://www.cbs.dtu.dk/services/TMHMM) was used to predict the orientation of the C-terminus of the protein. However, since PSEN2 fairly resembles to PSEN1
51
Figure 3.4. Interaction maps of human p24 proteins
MYTHS experiments were performed on the luminal domains of human p24 proteins. The p24 members are color-coded according to the family in which they belong to. NC = non-categorized. Only two interactions were detected (p24A and
TMP21). The interactions of the GOLD domain of p24A and TMP21 were also tested and the same results were obtained. A table is provided to distinguish the different names for a same p24 protein (Section 1.2.3).
52 gp25L
p26 α Systematic Name Alternate Names p27 TMED1 ST2L, IL1RL1LG, Tp24 β TMED2 p24A, RNP24 TMED3 p26, p24B
TMED6 γ TMED4 p25, GMP25, ERS25 TMP21 TMED5 p28, CGI-100 GOLD δ TMED6 NC p24A TMED7 CGI-109, gp27, p27 p28 NC GOLD TMED9 gp25L TMED10 TMP21, p23 Tp24 p25 (62.2% identity, as calculated by the EMBOSS::needle pairwise alignment algorithm at http://www.ebi.ac.uk/Tools/emboss/align/index.html), it is possible that these two proteins have the same topology. The TMHMM server was also used to compare the data from the literature and as shown in Table 3.4, all of them were in agreement except for PEN-2. The presence of a signal sequence was predicted by the signalP server (http://www.cbs.dtu.dk/services/SignalP). As an alternate method, the sequences were inserted into various pHYPER1 and pHYPER2 plasmids that contain a mutation (F980L) in the RNAse domain of
Ire1p, rendering it constitutively active (Figure 3.1C). The reporter would be activated if this domain was in the cytoplasm. This system was also used to verify the expression level of the proteins. As summarized in Table 3.4, most of the components were not expressed.
Table 3.4 Signal sequence, number of TMD span, and C-terminus orientation of -secretase components Hyperactive -secretase SignalP TMHMM Literature RNAse component Server Server System Cytosolic, 1 TMD Cytosolic, APP SS No expression (Haass, 2002) 1 TMD APPΔTMD Not found SS Luminal Luminal Cytosolic, 7 TMD Cytosolic APH-1 SS No expression (Fortna, 2004) 7 TMD Cytosolic, 1 TMD Cytosolic NCSTN SS No expression (Jutras, 2005) 1 TMD Luminal, 2 TMD Cytosolic, PEN-2 SS No expression (Bergman, 2004) 2 TMD Cytosolic, 8 TMD (Jutras, 2005) Luminal, PSEN1 No SS No expression Luminal, 9 TMD 9 TMD (Spasic, 2006) Luminal, PSEN2 Not found No SS No expression 9 TMD
54 Upon performing the MYTHS experiment, no interaction was detected between the -secretase components or with TMP21, probably due to the fact that the majority of them were not expressed. Thus, no conclusion could be drawn for the interactions among the -secretase components.
3.4 Random mutagenesis of Erp4G
Since the GOLD domain of Erp4p (Erp4G) interacts with that of both
Erp1p (Erp1G) and Erp6p (Erp6G, Figure 3.2C-D), I attempted to define the region of interaction of Erp4G with its partners by generating Erp4G mutants.
Erp4G was selected because it has two interactors and if an Erp4G mutant loses interaction with only one partner, it suggests that its structure is not compromised.
Mutations in Erp4G sequence were generated by PCR mutagenesis with manganese chloride. The PCR products were inserted into digested pMYTHS3-
SS vector by in vivo recombination in yeast. The advantage of this method is that it allows the generation of numerous colonies each containing a plasmid with potential different mutations of Erp4G in one step and that the colonies could be used immediately for MYTHS experiments. The latter was performed with the
Erp4G mutants as baits and Erp1G, Erp6G, and empty vector as preys. Three mutants that lost interaction with only one interactor have been generated; two that lost interaction with only Erp1G and one with Erp6G (Figure 3.5A).
Sequencing results revealed that the mutations were located at different regions of the Erp4G amino acid sequence (Figure 3.5C). The mutation of Erp4G (V51M) that lost interaction with Erp6G was in the middle of the GOLD domain whereas those of Erp4G (I114A) and Erp4G (T115P) that lost interaction with Erp1G were
55
Figure 3.5. Erp4G mutants generated by PCR random mutagenesis
Mutants of Erp4G were generated by PCR random mutagenesis followed by in vivo recombination and MYTHS experiments were performed to screen for mutants that lost interaction with either Erp1G or Erp6G.
A. MYTHS of the three Erp4G mutants generated by PCR random mutagenesis.
Two mutants have lost interaction with Erp1G and one with Erp6G.
B. Verification of protein expression level of the Erp4G mutants using the
hyperactive RNAse system. Expression levels were assessed based on cell
growth.
C. Alignments of the amino acid sequences of Erp4G mutants with wild type.
56
Erp1G Erp6G EV A I114A B
F4S, T115P I114A F4S, T115P V51M Erp4G WT
V51M
Erp4G WT
Signal Sequence (19 aa) Erp4G WT MRVFTLIAILFSSSLLTHAFSSNYAPVGISLPAFTKECLYYDLSSDKDVLVVSYQVLTGG C I114A MRVFTLIAILFSSSLLTHAFSSNYAPVGISLPAFTKECLYYDLSSDKDVLVVSYQVLTGG F4S, T115P MRVSTLIAILFSSSLLTHAFSSNYAPVGISLPAFTKECLYYDLSSDKDVLVVSYQVLTGG V51M MRVFTLIAILFSSSLLTHAFSSNYAPVGISLPAFTKECLYYDLSSDKDVLMVSYQVLTGG
Erp4G WT NFEIDFDITAPDGSVIVTERQKKHSDFLLKSFGIGKYTFCLSNNYGTSPKKVEITLEK I114A NFEIDFDITAPDGSVIVTERQKKHSDFLLKSFGIGKYTFCLSNNYGTSPKKVEATLEK F4S, T115P NFEIDFDITAPDGSVIVTERQKKHSDFLLKSFGIGKYTFCLSNNYGTSPKKVEIPLEK V51M NFEIDFDITAPDGSVIVTERQKKHSDFLLKSFGIGKYTFCLSNNYGTSPKKVEITLEK located at the end. In the case of the Erp4G (T115P) mutant, there was also a mutation in its signal sequence (F4S), but it was not believed that it would cause the loss of interaction with Erp1G.
The protein expression level of those mutants was verified using the hyperactive RNAse system (Figure 3.1C). It should be noticed that the expression level of all three mutants was decreased compared to the WT of Erp4G (Figure
3.5B). The reduction of expression and the loss of interaction with one partner may be explained by the weakness of interaction. If there was a mutation at the putative area of interaction and that the level of expression was decreased, then the interaction would be lost or decreased to the level that it could not be detected by MYTHS experiments. I then wondered if the Erp4G mutants would have gained any interaction with other p24 proteins than its interactors at WT condition.
To find out, I performed MYTHS experiments and the results were negative. I conclude that Erp1G and Erp6G interact with Erp4G at different regions of the latter and that these interactions might not be strong.
3.5 Yeast three-hybrid of Erp1G, Erp6G, and Erp4p
The locations of the mutated residues of the Erp4G mutants suggest that the interaction areas of Erp4G with Erp1G and Erp6G are different even though the level of protein expression is decreased in the mutants compared to the WT. I speculated that Erp4G might serve as an adaptor for the interaction between
Erp1G and Erp6G. However, since endogenous Erp4G did not seem to provide this bridging, overexpressing it exogenously might create this interaction. To test this hypothesis, a yeast 3-hybrid experiment was performed.
58 In order to ensure that endogenous Erp4p did not affect the bridging between Erp1G and Erp6G, I knocked out ERP4 in the strains Y574 and Y575 by replacing the gene with HygMX and KanMX cassettes respectively. The PCR products of HygMX and KanMX cassettes were first transformed separately into
EGY6 cells in which ERP4 was previously knockout by replacement with NatMX cassette (Garneau, 2004). A PCR was performed on the isolated choromosomal
DNA from EGY6 cells of which the replacement was successful and the product was transformed into Y574 or Y575. The extra step allowed the generation of longer sequences from ERP4 ORF flanking the cassettes in the PCR product, thereby rendering the knockout easier. Gene knockout was verified by restriction digestion on the ERP4 ORF PCR product from isolated chromosomal DNA of
Y574 and Y575 cells. The HygMX and KanMX cassettes had a HincII and
HindIII restriction site respectively. As a control, the PCR product of ERP4 was also digested with SalI and EcoRI as well as HincII and HindIII. As shown in
Figure 3.6A, the expected digest fragments were obtained; thus the knockout was successful. The Y574 and Y575 strains with an ERP4 deletion were termed ETY1 and ETY2 respectively.
The full length sequence of ERP4 was inserted into the pADH426 vector, a 2µ plasmid that has a constitutively active ADH promoter (Mumberg, 1995), so
ERP4 will be overexpressed. ETY1 was transformed with pMYTHS plasmids
(Erp1G and Erp6G) and ETY2 with pADH426-ERP4 and MYTHS experiments were performed with appropriate controls. The results showed that overexpression of Erp4p was able to create an interaction between Erp1G and Erp6G (Figure
3.6B). To find out if this interaction was real, cells were allowed to lose their
59 Figure 3.6. Yeast three-hybrid experiment of Erp1G, Erp6G, and Erp4p
A yeast three-hybrid experiment was performed to find out if Erp4p could bridge an interaction between Erp1G and Erp6G.
A. Verification of ERP4 KO in Y574 and Y574 cells by restriction digestion on
PCR products of ERP4 ORF of the chromosomal DNA. The expected
fragment sizes are indicated between brackets. Lane 1: 1kb ladder (Invitrogen).
Lane 2: ERP4 undigested (1668bp). Lane 3: ERP4 digested with HincII (173,
627, and 871bp). Lane 4: ERP4 digested with HindIII (1668bp). Lane 5:
HygMX undigested (2710bp). Lane 6: HygMX digested with EcoRI and SalI
(171, 456, 119, 885, and 985bp). Lane 7: HygMX digested with HincII (171,
969, and 1474bp). Lane 8: KanMX undigested (2490bp). Lane 9: KanMX
digested with EcoRI and SalI (119, 171, 456, and 1654bp). Lane 10: KanMX
digested with HindIII (1215 and 1185bp). Lane 11: ERP4 digested with EcoRI
and SalI (171, 456, and 1041bp).
B. Yeast three-hybrid MYTHS of Erp1G and Erp6G with Erp4p overexpressed
with pADH426 plasmid. Cells were grown on SD–LTUAi medium. The
legend in the grid indicates the plasmids in the cells. The order is always
pMYTHS3-SS, pMYTHS4-SS, and pADH426. EV = empty vector.
C. Plasmid loss experiment to determine the dependency of the cells to the
plasmid expressing Erp4p. Cells were grown on SD–ADE medium after
plasmid loss in SC medium. The asterisks indicate that cells still had all three
plasmids.
60 erp4Δ cells WT cells Erp1G- Erp6G- Erp1G- Erp6G- A C Erp1G- Erp6G- Erp1G- Erp6G- 1 2 3 4 5 6 7 8 9 10 11 EV EV EV EV
2036 * 1636 * * * * * * * * * * ** 1018 * * ** * * *** * * 506 * * * * * * * * * 396 * * ** * * *** ** * 201
Erp1G- Erp1G- Erp6G- Erp6G- Erp6G- Erp6G- Erp1G- Erp1G- Erp4p Erp4p Erp4p Erp4p B WT cells erp4Δ cells Erp1G- Erp6G- EV- EV- EV- EV- Erp1G- Erp6G- EV EV EV EV
Erp1G- Erp6G- EV- Ste11p- Erp6G- Erp1G- EV- Ste50p- EV EV EV EV
Erp1G- Erp6G- EV- EV- EV- EV- Erp1G- Erp6G- Erp4p Erp4p Erp4p Erp4p
Erp1G- Erp6G- EV- Ste11p- Erp6G- Erp1G- EV- Ste50p- Erp4p Erp4p Erp4p Erp4p plasmids in SC medium to determine the dependency of the cells to them. Cells were tested in SD-ADE medium and they should not have grown if they did not have all three plasmids containing p24s since the reporter should not be activated.
However, it seemed that the cells did not depend on the expression of Erp4p as the cells grew independently from the number of plasmids they contained (Figure
3.6C). Nevertheless, upon testing the frequency of loss of plasmids, pADH426-
ERP4 was the one that was lost the most often. Thus, it is not possible to clearly conclude that Erp4p serves as a bridge between Erp1G and Erp6G. Nevertheless, it is possible that since ERP4 is part of a 2µ plasmid, recombination might have occurred such that the ERP4 gene is lost, but the marker (URA3) was kept. This could be verified by isolating the plasmids and retransform them into yeast cells.
3.6 Co-overexpression of p24s in yeast
Since there were many p24-p24 interactions detected in MYTHS experiments and because there was no lethal phenotype in p24 deletion (Springer,
2000), I wanted to find out the outcome of p24 co-overexpression. This experiment was previously done only with ERP1, ERP2, EMP24, and ERV25 in all possible combinations (Garneau, 2004). I constructed the plasmids for the remaining p24s (ERP3-ERP6). After obtaining the transformants for every combination in W303-1A cells, a co-overexpression serial dilution screen (Figure
3.7A) was performed and a heat map for a plate of 5000 cells incubated for 72 hours was generated to facilitate the analysis (Figure 3.7B). This plate was chosen because it reflected the growth differences the best. There were four combinations in which the cells exhibited a major growth defect when the p24s were
62 overexpressed (ERP1-ERP1, ERP2-EMP24, EMP24-EMP24, and ERV25-ERV25) with ERP2-EMP24 being the only heterogeneous set. These results were in agreement with those observed previously (Garneau, 2004). The majority of combinations grew just like the double empty vector (EV-EV) control especially those combining from ERP3 to ERP6. Others exhibited slower growth especially those combining with ERP1, EMP24 or ERV25, which was not surprising considering the fact that they had a growth defect even when singly expressed.
Growth curves were done on the combinations that did not grow in the screen to determine their doubling time. It is more accurate to look at the growth profile by measuring the OD600 in a culture of bigger volume (5mL) than that of 200µL from a plate. The growth rate of all combinations was reduced compared to the EV-EV control (Figure 3.7C). EMP24-EMP24 was the culture that grew the slowest with an estimated doubling of 35 hours followed by ERV25-ERV25 (Figure 3.7D).
To test whether the cells were growth arrested or dead for those that co- overexpressed p24s and that did not grow in the screen, aliquots of 5000 cells were cultured on galactose to induce expression and plated on dextrose after 24,
48, 72, and 96 hours of induction to monitor their recovery. There were a small percentage of cells that survived the co-overexpression of p24s (6% or less), except for ERP2-EMP24, which seemed to have reverted the phenotype after four days of induction on galactose (Figure 3.8A-B).
Besides the growth defect phenotype, co-overexpression of some p24s also exhibited increase in cell size compared to the EV-EV control as analyzed by brightfield microscopy. A heat map was also generated to compare cell size of every combination (Figure 3.9B). The majority had the same cell size as the
63 double empty vector control, which was about 5 to 7 in diameter (Figure 3.9A).
EMP24-EMP24 (Figure 3.9A) and ERV25-ERV25 were those that showed the biggest increase in cell size, to about 10 to 12 for the largest cells. ERP1-ERP1 and ERP2-EMP24 cells had a cell size increase to about 8 to 10 for the biggest cells. Surprisingly, some combinations also displayed a cell increase even though they were growing (ERP1-ERP4, ERP3-ERP3, ERP4-ERP4, ERP6-ERP6, and
ERP6-EMP24). Their size increase was also in range of 8 to 10.
To verify whether the cell size increase of some combinations affected any organelles, fluorescence microscopy experiments were performed. The cells were stained with Hoechst 33258 to verify the nuclei, Calcofluor White to check the bud scars, Rhodamine 123 for the mitochondria, and FUN-1 for the cylindrical intravacuolar structures (CIVs). The nuclei seemed to be normal as there were no multiple nuclei or any other aberration that could be detected. The integrity of the mitochondria did not seem to be compromised either. There were differences with
FUN-1 dye for ERP1-ERP1, ERP2-EMP24, EMP24-EMP24, and ERV25-ERV25 as some CIVs were not stained as well and there was some dye that remained in the cytoplasm compared to the EV-EV control. FUN-1 is a fluorescent dye that is green when present in the cytoplasm, but turns orange-red when transported into the vacuole where it stains the CIVs. For the transport to occur, the integrity of the
PM and the cell metabolic activity must not be compromised (Millard, 1997).
Since there was a problem for FUN-1 to stain CIVs, the viability of the cells was likely to be affected. For Calcofluor White staining, there were less bud scars for the same combinations than the EV-EV control, which was consistent with the
64
Figure 3.7. Growth phenotype of W303-1A cells that co-overexpressed p24 proteins.
W303-1A cells were induced on 4% galactose to double overexpress p24 proteins in a serial dilution screen. The cell growth was assessed based on OD600 measurements.
A. Example of a plate from the serial dilution screen
B. Heat map of the growth phenotype based on OD600 values. This plate
contained 5000 cells per well and was incubated at 30oC for 72 hours.
C. Growth curves for cells of the four combinations that did not grow in the
screen. Cells were induced in 5mL selective medium containing 4% galactose
for a more accurate growth profile.
D. Estimated doubling time from growth curves in B.
65 A C 2 1,8 1,6 1,4 1,2 1 OD600 0,8 ERP1-ERP1 0,6 ERP2-EMP24 0,4 EMP24-EMP24 ERV25-ERV25 0,2 EV-EV 0 p426
p425 ERP1 ERP2 ERP3 ERP4 ERP5 ERP6 EMP24 ERV25 EV 0 50 100 150 B EV ERP1 Time (h) ERP2 ERP3 OD600 ERP4 >0.800 ERP5 Combinations DT(h) ERP6 D >0.5-0.8 ERP1-ERP1 10.5 >0.2-0.5 EMP24 >0.1-0.2 ERV25 ERP2-EMP24 15 0-0.1 EMP24-EMP24 35 ERV25-ERV25 25 ERP1 ERP2 ERP3 ERP4 ERP5 ERP6 EMP24 ERV25 EV p425 EV-EV 4.5 p426
Figure 3.8. Recovery on dextrose after induction on galactose for the cells that did not grow when co-overexpressing a subset of p24s in the serial dilution screen
Aliquots of 5000 W303-1A cells were induced in liquid selective medium containing 4% galactose to double overexpress p24 proteins for different periods of time. Induction was then repressed by plating the cells onto selective medium containing dextrose. The percent survivors were assessed after two days of incubation in dextrose medium.
A. Table displaying the percentage of survivors from 5000 cells for each
combination after different periods of induction.
B. Graphical representation of the percent survivors from 5000 cells for each
combination after different periods of induction.
67 Time (h) ERP1-ERP1 ERP2-EMP24 EMP24-EMP24 ERV25-ERV25 EV-EV A 0 100 100 100 100 100 24 0,32 2,78 0,84 0,58 100 48 2,34 5,18 0,22 0 100 72 5,52 7,06 0,06 0,3 100 96 5,94 100 0,02 0 100
B 100 80 ERP1-ERP1 60 ERP2-EMP24 EMP24-EMP24 40 ERV25-ERV25 EV-EV
Percent Survivors Percent 20 0 1 2 3 4 Days
Figure 3.9. Cell size phenotype of W303-1A cells that co-overexpressed p24 proteins.
W303-1A cells were induced on 4% galactose to double overexpress p24 proteins.
The cell size was assessed by brightfield microscopy.
A. An example (EMP24-EMP24 and EV-EV) of the cell size phenotype by
brightfield microscopy.
B. Heat map of the cell size phenotype
.
69 A
EV-EV EMP24-EMP24
Cell Size p426
p425 ERP1 ERP2 ERP3 ERP4 ERP5 ERP6 EMP24 ERV25 EV >> EV-EV(>10-12µ) B EV > EV-EV (~7-10µ) ERP1 = EV-EV (~6µ) ERP2 ERP3 ERP4 ERP5 ERP6 ERP1 ERP2 ERP3 ERP4 ERP5 ERP6 EMP24 ERV25 EV EMP24 p425 ERV25 p426
Figure 3.10. Phenotype of W303-1A cells that co-overexpressed ERV25-
ERV25 stained with Calcofluor White
Cells overexpressing p24s that had a growth defect or a cell size phenotype were subjected to staining by fluorescent dyes to verify the integrity of various organelles and cellular functions. Here, ERV25-ERV25 cells stained with
Calcofluor White had an extra phenotype.
A. Brightfield image of cells that co-overexpressed EV-EV
B. Brightfield image of cells that co-overexpressed EV-EV stained with
Calcofluor White
C. Fluorescence image of cells that co-overexpressed EV-EV stained with
Calcofluor White visualized under the DAPI filter
D. Brightfield image of cells that co-overexpressed ERV25-ERV25
E. Brightfield image of cells that co-overexpressed ERV25-ERV25 stained with
Calcofluor White
F. Fluorescence image of cells that co-overexpressed ERV25-ERV25 stained with
Calcofluor White visualized under the DAPI filter
71 A EV-EV B EV-EV C EV-EV
ERV25-ERV25 D E ERV25-ERV25 F ERV25-ERV25 fact that the cells bearing those combinations were not dividing. Moreover,
Calcofluor White seemed to have an effect on the cells that double overexpressed
ERV25 as large cells with an abnormal shape were observed (Figure 3.10E-F).
To conclude, p24 co-overexpression is harmful for a subset of combinations, as cells do not recover after removing them from induction medium.
Moreover, it did not affect the integrity of the nucleus or the mitochondria even if cell viability seems to be affected for a subset of combinations. The large cell phenotype and the phenotype shown by ERV25-ERV25 cells stained with
Calcofluor White suggest that the p24s proteins might be involved in cell cycle or cell wall maintenance pathways (Section 4.3).
3.7 Expression of recombinant p24 proteins
In order to study the in vitro physical interactions of p24s and as a mean to validate the MYTHS results, insect Sf9 cells were used to express the p24 proteins. The yeast and human p24s were previously expressed in bacterial E.coli cells, but were insoluble. As a solution, the baculovirus-insect cell system was considered because it might help in improving the solubility of the p24s since insect cells could perform the eukaryotic post-translational modifications that the proteins might require. Based on the MYTHS results, the luminal sequences of human p24A and TMP21, and those of yeast ERP1, ERP4, and ERP6 both luminal and GOLD domains, were selected for insertion into the pFASTBac-Cpo-
His6 plasmid. This vector contains a signal sequence that fuses to the N-terminus of the protein of interest and the sequence will trigger secretion into the medium if the protein is soluble. The protein also bears a C-terminal His6-tag for purification
73 purposes. The protein solubility was assessed based to the ability of the Sf9 cells to secrete it into the medium. Although the majority of the proteins were expressed except for Erp4G-His6, they were not soluble as they were detected in the pellet by Western blotting for anti-His (Figure 3.11A). Only TMP21L-His6 was soluble since it was found in the medium. However, in a large-scale expression and purification, although it was detected by Western blotting,
TMP21L-His6 was not visible by Coomassie staining (Figure 3.11B); thus the expression level was low. Considering this issue, another strategy was examined.
Since there were many interactions detected in MYTHS experiments, I hypothesized that a p24 protein might need its interactors to remain soluble and stable when expressed. Therefore, I decided to coexpress the GOLD domains of the following pairs in E.coli: ERP1-ERP4, ERP4-ERP6, ERP3-ERP5, and
EMP24-ERV25 in addition to the luminal domains of TMP21-p24A (TMP21L- p24AL). The sequences of the pairs were inserted into the pRSFDuet-I plasmid, which has two MCS. The first protein has an N-terminal His6-tag and the second a
C-terminal S-Tag (Kim, 1993). Only the combination Erp1G-Erp4G and
TMP21L-p24AL were expressed and soluble when verified by Western blotting for anti-His and anti-S-Tag (Novagen), but the former was not visible on a
Coomassie stain gel. In the case of TMP21L-p24AL (Figure 3.12A), MS results revealed that His6-TMP21L (20.2kDa) was abundantly expressed whereas only traces of p24AL-S (19.6kDa) could be detected. I then thought that perhaps switching the tags could help in p24AL expression. However, upon testing it,
TMP21L-S was not expressed. Thus, I concluded that the plasmid second ORF was not expressed. I then decided to coexpress both proteins in two compatible
74 plasmids (pRSFDuet-I and pETDuet-modified). In this case, one protein would have an N-terminal His6-tag and one with His8-tag. His8-p24AL was expressed at slightly higher levels, but was insoluble. After further tests, I found out that His6-
TMP21L was expressed and soluble without the presence p24AL. Thus, His6-
TMP21L was used for subsequent purification.
His6-TMP21L was first purified manually using Ni-NTA beads (Figure
3.12B). Upon dialysis to rid the protein solution of imidazole, an anion-exchange by Mono Q chromatography was used to discard the DnaK chaperone (70kDa) identified by MS (Figure 3.12C). Finally, a gel filtration step was performed to rid the protein solution of any remaining impurities (Figure 3.12D). A single band at
20.2kDa could be seen after three purification steps with a yield of 2.5mg/L. The pure protein could be safely concentrated down to 30 mg/mL. I conclude that I succeeded in purifying soluble recombinant His6-TMP21L in relatively high yield and it could be used for subsequent characterization experiments.
3.8 NMR, CD, crystallization screen, and pulldown assay for His6-TMP21L
To verify whether the protein was folded, a NMR for 1H experiment was performed and the acquired spectrum of His6-TMP21L revealed that it is mostly unfolded or unstructured. Circular dichroism spectra revealed it did not have any consistent secondary structures. A crystallization screen was set up using two different protein concentrations (15 and 30 mg/mL) with four suites of 96 conditions. Since the protein was unstructured, the protein probably would never crystallize. If His6-TMP21L was unfolded, then it should bind to chaperones to help in its folding if a pull-down experiment using rat smooth ER was done.
75 However, such was not the case as no protein could be identified by silver stain or
MS for pulldown with His6-TMP21L-coated beads.
76
Figure 3.11. Expression of yeast and human p24 proteins in Sf9 insect cells
Sf9 insect cells were infected with baculovirus that encode various fragments of p24 proteins. The solubility of the p24s was assessed based on the ability of the insect cells to secrete them into the medium.
A. Western blotting with anti-His probe antibody to verify the expression and
solubility of various fragments of p24 proteins in Sf9 cells. P = pellet, M=
medium
B. Coomassie Blue staining and Western blot (anti-His probe) of a large-scale
expression and purification of TMP21L-His6 (21kDa). Lane 1: Molecular
weight markers (Biorad). Lane 2: medium in which TMP21L-His6 was
secreted. Lanes 3-4: Flow through from Ni-NTA beads column. Lanes 5-6:
Washes. Lanes 7-14: Elution fractions. Lane 15: Ni-NTA beads after
purification.
77 P M B A Erp1G-His 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Erp1L-His6
Erp4G-His6
Erp4L-His6
Erp6G-His6 20kDa
Erp6L-His6
P M α-His6
TMP21L-His6
p24AL-His6
Figure 3.12. Expression tests and large-scale coexpression and purification of
His6-TMP21L
The luminal domain of TMP21 was coexpressed with that of its interacting partner p24A according to MYTHS results. TMP21 has a His6-tag at its N- terminus (denoted His6-TMP21L) and p24A has an S-Tag at its C-terminus
(denoted p24AL-S). His6-TMP21L has a calculated molecular weight of 20.2kDa whereas that of p24AL-S is 19.6 kDa. Black arrow indicates the expected band.
A. SDS-PAGE followed by Coomassie Blue staining of a coexpression and
solubility tests of His6-TMP21L and p24AL-S. Lane 1: Molecular weight
markers (Biorad). Lane 2: Uninduced culture. Lane 3: Induced culture. Lane
4: Pellet. Lane 5: Supernatant.
B. Coomassie Blue staining of Ni-NTA Purification of His6-TMP21L. A sample
of each step of the purification procedure was resolved by SDS-PAGE. Lane
1: Molecular weight markers (Biorad). Lane 2: Uninduced culture. Lane 3:
Induced culture. Lane 4: First pellet. Lane 5: Second pellet. Lane 6: Lysate.
Lane 7: Flow through. Lanes 8-9: Washes. Lanes 10-14: Elution fractions.
Lane 15: Ni-NTA beads after purification.
C. Coomassie Blue staining of a mono Q chromatography of His6-TMP21L.
Lanes 8-24: Elution fractions that contained His6-TMP21L
D. Coomassie Blue staining of a gel filtration of His6-TMP21L. Lanes 8-20:
Elution fractions that contained His6-TMP21L
79 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 A 1 2 3 4 5 B
20kDa 20kDa
C 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 D 8 9 10 11 12 13 14 15 16 17 18 19 20
Chapter 4
Discussion
81 4.1 Interactions between yeast p24 proteins
The p24 proteins were shown to form complexes among them and their stability depends on each other (Fullekrug, 1999; Marzioch 1999). However, the pairwise interactions among p24s have not been demonstrated except in large- scale screens in which only the interactions of full p24s were investigated (Miller,
2005; Schuldiner, 2005; Krugan, 2006; Tarassov 2008). In this study, I have determined the interactions for different domains of both yeast and human p24 proteins by MYTHS (Figure 3.1A), and I conclude that there is some specificity among p24 interactions and these occur mostly through the GOLD domain with some contribution from the DOG sequence (Figure 3.3D). At first glance, it seems that the results of this study are contradictory with those found in the literature
(Ciufo, 2000). Indeed, the authors of this article have shown that it was the heptad repeat that forms coiled coil motifs N-terminal of the TMD and downstream of the GOLD domain of the protein that was responsible for the interaction among p24s (Ciufo, 2000). The authors have shown that replacement of the heptad repeat of Emp24p by that of Erv25p to generate an Emp24p-Erv25p chimera failed to stabilize Erv25p in Δemp24 cells. In this study, I observed that Erv25D, which corresponded to the sequence of the Erv25p heptad repeat, indeed did not interact with itself (Figure 3.3A). Ciufo and Boyd have also shown that replacement of
Erv25p heptad repeat by that of Emp24p (Erv25p-Emp24p chimera) in Δemp24 cells succeeded in stabilizing slightly Erv25p. I have also observed that Emp24D, which corresponded to the heptad repeat of Emp24p, interacted with Erv25L or
Erv25G, but the interaction was not as strong as that of Emp24G-Erv25G or
Emp24L-Erv25L. Thus, Emp24G may be the main interacting domain with
82 Erv25G and that Emp24D contributes to strengthen this interaction. However, in this study, the self-interactions of Erv25G and Erv25L were detected; thus, they must be stable. To explain this discrepancy, it is possible that endogenous
Emp24p or other p24s contributed to the stabilization of Erv25L and Erv25G. The contribution from other p24s seemed to be not the case as Erv25p was not stabilized in cells in which only EMP24 was knocked out (Marzioch, 1999; Ciufo
2000). However, to exclude the possibility, the determination of p24 interactions by MYTHS using strains in which all eight members are knocked out would be desirable. This is achievable since p24 proteins are not essential in yeast and an eight-fold deletion strain grows like the WT (Springer, 2000). Nevertheless, Ciufo and Boyd have only tested Emp24p and Erv25p and despite some differences in terms of domain interactions among p24 proteins in this study, it is still true the majority of them involve the GOLD domain with contribution from the DOG sequence (heptad repeat).
Immunoprecipitation studies have demonstrated that Erp1p, Erp2p,
Emp24p, and Erv25p form a complex (Marzioch, 1999). In this study, Erp1p indeed interacts with Erp2p and Emp24p with Erv25p for both the luminal and
GOLD domains (Figure 3.2B-D). However, Erp1p and Erv25p interacts only when their whole luminal domain is present (Figure 3.3B). Upon further testing, I found out that it is Erp1D that is responsible for the interaction with Erv25G
(Table 3.3). As a simplistic view, I propose that the GOLD domain of Erp1p interacts with that of Erp2p and the DOG sequence of Erp1p interacts with the
GOLD domain of Erv25p. Finally, the GOLD domain of Emp24p interacts with that of Erv25p (probably at a region different from Erp1D) with contribution from
83 the DOG sequence of Emp24p. Therefore, Erp1p-Erp2p and Emp24p-Erv25p complexes are linked together by the interaction of the DOG sequence of Erp1p and the GOLD domain of Erv25p. Again, I cannot rule out the possibility that endogenous p24s contribute to the interactions and more work is needed to verify this hypothesis.
Other studies have shown that Erp1p and Erp2p were not required for incorporation of Emp24p and Erv25p into COPII vesicles (Belden, 2001b). It is possible that the interaction of the former with the latter is transient or weak depending on the situation. For example, Erp1p and Erp2p perhaps only associate with Emp24p and Erv25p to package specific cargo proteins, which are yet to be identified, into COPII vesicles. I would suggest that the interaction of the putative cargo protein is likely to occur through the DOG sequence of Erp2p since Erp1p and Erp2p interact with each other through their GOLD domain.
It is interesting to note that there were many interactions detected by
MYTHS among the remaining of p24 proteins (Erp3p-Erp6p), but they do not seem to have any apparent function in the cell except the fact that they can substitute for members of the same subfamily (Marzioch, 1999). For example,
Erp5p and Erp6p can substitute for Erp1p function in Δerp1 cells (Marzioch,
1999). In this study, members of the same family do not interact with each other through their GOLD domain except for the self-interactions and have common p24 interactors (Figure 3.2C-D). Thus, it is possible that these p24s with an unknown function are present in the cell for helping other members with more predominant functions, but this idea remains to be tested. However, this was
84 shown in mammalian cells as p25, and p26 can increase the transport efficiency by p23 and p24, but the former cannot substitute for the latter (Emery, 2000).
Although the interactions were clear in the MYTHS experiments, I cannot rule out the fact that endogenous p24s might have contributed because the strains contained all the eight wild type members. At least for the case between Erp1G and Erp6G, which do not interact (Figure 3.2C-D), endogenous Erp4p does not have an effect on their interaction (Figure 3.6B). Exogenously overexpressed
Erp4p may or may not bridge the interaction between Erp1G and Erp6G whether endogenous Erp4p was present or not. It was not possible to validate the seemingly positive Y3H results (Figure 3.6B) as plasmid loss experiments were not conclusive (Figure 3.6C).
From random mutagenesis experiments, Erp4G interacts with Erp1G and
Erp6G at different parts of its GOLD domain. However, the expression level of the Erp4G mutants is decreased compared to the WT (Figure 3.5B). The loss of interaction of Erp4G with either Erp1G or Erp6G is likely not due to the nature of the interaction as the WT and mutated residues were all hydrophobic. Thus, the interactions are potentially weak and when the expression level is decreased added by mutation on the putative interaction area, the interaction would be just lost or become undetectable.
The solved structure of human SEC14L2 protein is the only one that contains a GOLD domain (Stocker, 2002). This protein, also known as the supernatant protein factor (SPF), stimulates the activity of squalene epoxidase in the cholesterol biosynthesis pathway (Shibata, 2001). Its GOLD domain consists of seven -strands (Stocker, 2002). Although an alignment by T-COFFEE
85 (Notredame, 2000) exhibits significant conserved residues between the GOLD domains of p24 proteins of different species, including yeast and human
(Anantharaman, 2002), it was not advisable to use the SEC14L2 structure to map the mutations of Erp4G or to do accurate modeling as the sequence identity and similarity between the two GOLD domains were only 21% and 35% respectively.
The random mutagenesis strategy could also be used to define the interactions between other p24s such as those of Erp5p and Emp24p with Erv25p.
Defining the area of interaction of Emp24p and Erv25p would be of interest since they are the homologs of human p24A and TMP21 respectively (Dominguez,
1998), and TMP21 is involved in AD. Since the components of the γ-secretase were not expressed in this study, defining the residues responsible for the interaction between Emp24p and Erv25p would allow site-directed mutagenesis on p24A and TMP21, which in turn, would allow the determination of the residues of interaction between these two proteins. This is achievable since p24A and TMP21 have more than 50% sequence similarity with their yeast counterparts.
Alternatively, other interactors of p24A or TMP21 could be used, such as
GRASP55 and GRASP65 (Barr, 2001), two Golgi membrane proteins, if they are expressed in yeast even though they would be out of their natural environment.
The MYTHS was a good system in this study because it uses the ER- resident Ire1p; thus, fusing the p24 proteins to the TMD/kinase/RNAse domains of Ire1p does not put them out of context. Other systems such as split-ubiquitin
Y2H (Miller, 2005) and protein-fragment complementation assay (PCA, Tarassov,
2008), which is a modified split-ubiquitin system that uses dihydrofolate reductase (DHFR), use fusion of two halves of a reporter protein to the C-
86 terminus of two proteins of interest. If these two proteins interact, the two halves will reconstitute into their native conformation. However, since ubiquitin and
DHFR are cytosolic proteins, fusion might disturb the topology of membrane proteins and cause mislocalization. This is particularly true for the p24 proteins because the cytoplasmic tail contains motifs for incorporation into COPI and
COPII vesicles (Dominguez, 1998; Belden, 2001b), and if the motifs are mutated, the p24s can be mislocalized (Nakamura, 1998; Barr, 2001; Emery, 2003). In the case of the full length p24s, the cytoplasmic tail is fused to a TMD made of up leucine residues (pMYTHS5 and pMYTHS6 plasmids), which is followed by
Ire1p TMD/kinase/RNAse domains. The p24 cytoplasmic tail is still in the cytosol, but the FF and KKXX motifs are out of position and since Ire1p has an ER targeting signal, it is unlikely that they would be packaged into COPII vesicles for transport. Furthermore, the TMD of p24 proteins contains a highly conserved glutamic acid residue and a 100% conserved glutamine residue (Fiedler, 1997). A charged amino acid in the TMD serves as a retention signal that is modulated by other TMD residues and FF motif in the cytoplasmic tail (Fiedler, 1997). Thus, it is possible that the localization of the p24s from all three methods is correct.
4.2 Human p24s and γ-secretase
There were only two interactions detected among human p24s (Figure 3.4), which is somewhat surprising because mammalian p24s were also shown to form hetero-complexes (Fullekrug, 1999). However, they were expressed in their natural environment whereas they were in yeast in this study. The expression level of human p24 proteins is probably lower in yeast rendering the interactions
87 undetectable. Such was not the case for p24A and TMP21 probably because they have a higher similarity with yeast p24s (Emp24p and Erv25p) and endogenous yeast p24s might also interact with these two human p24s.
Since the γ-secretase components were not expressed in yeast using pMYTHS plasmids, another method should be considered to determine their interactions. However, individually expressed components could be unstable. It would have been interesting to perform MYTHS experiments with the yeast homologs of the complex. However, yeast S.cerevisiae does not have endogenous
γ-secretase activity and no homolog of the components have been identified
(Edbauer, 2003). Interestingly, Edbauer and colleagues have shown that mammalian γ-secretase activity could be reconstituted in yeast and they concluded that the basic four components of presenilin, nicastrin, PEN-2, and APH-1 strictly constitute the active enzyme complex. Recently, Winkler and co-workers have succeeded in purifying an active form of the γ-secretase from HEK293 cells and they have demonstrated that TMP21 was not part of the active complex (Winkler,
2009). Other proteins that co-purified with the γ-secretase have also been identified and p24A, p25, and p26 were among them. Thus, even though these p24s are not part of the active complex, they might play a role in modulation of its activity like TMP21 does. Perhaps, different p24s interact with different members of the γ-secretase, but this hypothesis remains to be proven.
The uncharacterized YKL100C non-essential yeast gene is similar to a signal peptidase that has presenilin-like activity (Weihofen, 2002). It is probable that this gene is a homolog of some component of the mammalian γ-secretase, likely to be more presenilin (11% sequence identity) than nicastrin (7%), APH-1
88 or PEN-2. Interestingly, YKL100C interacts genetically with α-synuclein, a protein when mutated is responsible for the pathogenesis of the Parkinson’s disease (Willingham, 2003). Thus, there might be yeast genes that encode proteins involved in neurodegenerative diseases and they could be investigated to perhaps understand better the function of the homologous genes in human.
4.3 Co-overexpression of p24 proteins in yeast
Co-overpression of p24s has a harmful effect to a subset of combinations.
Indeed, cells overexpressing ERP1-ERP1, ERP2-EMP24, EMP24-EMP24, and
ERV25-ERV25 exhibit cell growth defect (Figure 3.7B) and this is not due to growth arrest since repression of induction does not recover growth except for
ERP2-EMP24 after a longer period of time (Figure 3.8). Overexpression of these pairs of p24s probably causes an imbalance in the transport of putative essential cargo proteins for which they are responsible. ERP2-EMP24 is able to revert the phenotype probably because since it is heterogeneous, less of the same p24 is expressed and the cell would a way to relieve the stress of p24 overexpression.
Co-overexpression causes a cell size increase effect for a subset of combinations, which include not only those with a growth defect, but also some that grow at or near control rate (ERP1-ERP4, ERP3-ERP3, ERP4-ERP4, ERP6-
ERP6, and ERP6-EMP24). The size increase can be as dramatic as the double of a normal cell, from 6 to 12 in diameter (Figure 3.9A). The cell size increase phenotype due to protein overexpression is not novel as co-overexpression of
Mst27p and Prm8p also leads to this aberration (Sandmann, 2003). Mst27p
(Multicopy suppressor of SEC21) is an integral membrane protein involved in
89 vesicle formation and Prm8p is a pheromone-related transmembrane protein that contains a FF motif involved in COPII binding (Sandmann, 2003). The cells co- overexpressing Mst27p and Prm8p grow at wild type rate and have a large vacuole, but the proteins are not degraded in there. Rather, they are retained in the
ER. Perhaps, co-overexpressed p24s are also retained in the ER due to the fact that they did not have the proper stoichiometry to exit the ER, which have previously proposed by other researchers (Emery, 2000; Rojo, 2000). Other studies have shown that overexpression of p24 proteins cause an enlargement of
ER (Blum, 1999; Rojo 2000). Since there is evidence that p24s have a structural role on the organelles of the early secretory pathway and that they are dependent on each other for proper cell localization, ER enlargement due to overexpression would not be surprising if it was in the case in this study. Thus, fusion of p24s to green fluorescent protein (GFP) would be a good strategy to use to localize the overexpressed p24s. Immunofluorescence experiments with markers such as calnexin for ER and GM130 for Golgi would be an option to consider in order to determine the integrity of these organelles. In this study, cells co-overexpressing p24s had a normal nucleus and mitochondria.
Sandmann et al have demonstrated that Mst27p and Prm8p do not interact and that overexpression of Mst27p or Prm8p with Sec20p, a v-SNARE protein, does not lead to the large cell phenotype (Sandmann, 2003). However, the authors did not specify if Sec20p interacts with Mst27p or Prm8p. In this study, it is not really possible to clearly conclude whether the phenotype is due to the non- interaction of the overexpressed pairs because most of them consist of double overexpression of the same p24, for which some self-interacts and some does not
90 (Figure 3.2B-D). As for the heterogeneous pairs (ERP1-ERP4, ERP2-EMP24, and
ERP6-EMP24), only the first pair interacts from MYTHS results. Thus, more work is still required to determine the cause of the phenotype.
Cells co-overexpressing p24s also have a large or fragmented vacuole. The large vacuole phenotype was also observed in co-overexpression of Mst27p and
Prm8p (Sandman, 2003). Several genes that have a vacuolar function interact genetically with p24s in a synthetic genetic array (SGA) screen (Garneau 2004,
Table 4.1). Thus, transport of those proteins might be affected in cells overexpressing some p24s, but remains to be proven.
Table 4.1 Genes involved in vacuolar function and genetic interactors with p24s p24 interactor Gene Functions (from http://www.yeastgenome.org) (Garneau, 2004) Component of the vacuole SNARE complex involved in vacuolar morphogenesis; functions VAM17 EMP24 with a syntaxin homolog Vamp3p in vacuolar protein trafficking Dubious ORF, unlikely to encode a protein; not conserved in closely related Saccharomyces VPS63 EMP24 species; 98% of ORF overlaps the verified gene YPT6; deletion causes a vacuolar sorting defect Protein of unknown function; heterooligomeric or homooligomeric complex; peripherally associated VPS13 EMP24 with membranes; involved in sporulation, vacuolar protein sorting and protein-Golgi retention Peripheral membrane protein with a role in endocytosis and vacuole integrity, interacts with MON2 EMP24, ERV25 Arl1p and localizes to the endosome; member of the Sec7p family of proteins
Cell size homeostasis in S. cerevisiae requires the cell to grow at critical size before it is allowed to undergo cell division (reviewed by Jorgensen, 2004).
Cell growth and cell cycle are coordinated; and there are numerous pathways that
91 contribute to regulate these processes. The large cell phenotype seen in co- overexpression of p24s could be due to a problem in these processes. Upon searching in databases, I could not find any physical interactor involved in cell cycle progression or division that interacts with any of the p24s except for Far8p, which is involved in G1 cell cycle arrest in response to pheromone. Far8p was detected to be interacting with Erv25p by phenotypic enhancement in a large- scale screen (Schuldiner, 2005). Since the haploid cells were grown separately from the other mating type, it is unlikely that Far8p is involved in the phenotype.
Since cells are not constitutively undergoing cell division, the proteins involved in the process are not always expressed (Jorgensen, 2004). Perhaps the p24s proteins interact with some of them, but these interactions could not be detected by large-scale systematic screens. A budding defect could also be the cause of the large cell phenotype. For example, the transport of Axl2p, which is an integral membrane protein found at the bud neck and is required for axial budding in haploid cells (Powers, 1998), might be affected because the function of its receptor for its incorporation into COPII vesicles, Erv14p, might be compromised. ERV14 interacts genetically with EMP24 and ERV25 (Garneau,
2004). If EMP24 or ERV25 is overexpressed, there might be effect on ERV14 as well. There are other p24 genetic interactors that are involved in cell maintenance
(Garneau, 2004). A list of these genes is provided in Table 4.2 and any of them could be affected in p24 co-overexpression, but this remains to be demonstrated.
Since there is increasing evidence that p24 proteins are involved in transport of GPI-anchored proteins (Castillon, 2008; Takida, 2008), the large cell phenotype might be caused by problems in transport of this type of proteins
92 Table 4.2 Genetic p24 interactors involved in cell wall maintenance Gene p24 interactor Functions (from http://www.yeastgenome.org) (Garneau, 2004) Serine/threonine MAP kinase involved in regulating the maintenance of cell wall integrity SLT2 ERV25 and progression through the cell cycle; regulated by the PKC-1 mediated signaling pathway Required for proper cell fusion and cell morphology; functions in a complex with Kel2p KEL1 ERP5 to negatively regulate mitotic exit; interacts with Tem1p and Lte1p; localized to regions of polarized growth; potential Cdc28p substrate Putative metalloprotease with similarity to the ECM14 ERP5 zinc carboxypeptidase family; required for normal cell wall assembly Sensor-transducer of the stress-activated PKC1- MPK1 kinase pathway involved in maintenance SLG1 EMP24 of cell wall integrity; involved in organization of the actin cytoskeleton; secretory pathway Wsc1p is required for the arrest of secretion response ERP1, ERP2, β-1,3-glucanosyltransferase; required for cell GAS1 EMP24, ERV25 wall assembly; cell surface protein
implicated in cell wall maintenance or cell cycle. A non-exclusive list of GPI- anchored proteins involved in these pathways is provided in Table 4.3. Future work is required to determine if the p24 proteins are involved in their transport and other GPI-anchored proteins involved in the processes.
Calcofluor White staining revealed that there were less bud scars for the combination of p24s that exhibited a growth defect, thus less cell division.
However, staining of ERV25-ERV25 cells led to an extra phenotype, which was further enlargement of cells and complete staining with the dye (Figure 3.10F).
Calcofluor White is an antifungal fluorescent dye that binds to chitin and increases chitin synthesis. However, the chitin synthesized in the presence of the
93 dye is abnormal leading to a weakened cell wall (Delom, 2006). Since Erv25p is postulated to be implicated in transport of cargo with increasing evidence for
Table 4.3 GPI-anchored proteins involved in cell cyle or cell wall maintenance Protein Functions (http://www.yeastgenome.org) Ecm33p Unknown function; has possible role in apical bud growth Ccw14p Covalently linked cell wall glycoprotein Cell wall protein, expression is periodic and decreases in response Tos6p to ergosterol perturbation or upon entry into stationary phase; depletion increases resistance to lactic acid Cell wall protein; upregulated by activation of the cell integrity Pst1p pathway, as mediated by Rlm1p; upregulated by cell wall damage via disruption of FKS1 Cell wall protein that functions in the transfer of chitin to β-1,6- Crh1 glucan; localizes to sites of polarized growth; expression is induced under cell wall stress conditions Cell wall protein that functions in transfer of chitin to β-1,6- Crh2p glucan; putative chitin transglycosidase; localized to the bud neck; has a role in cell wall maintenance Cell wall mannoprotein, linked to β-1,3- and β-1,6-glucan Cwp1p heteropolymer through a phosphodiester bond; involved in cell wall organization Cell wall glycoprotein; major constituent of the cell wall; plays a Cwp2p role in stabilizing the cell wall; involved in low pH resistance β-1,3-glucanosyltransferase; required for cell wall assembly; cell Gas1p surface protein Integral PM protein involved in the synthesis of the GPI core Gpi7p structure; mutations affect cell wall integrity
GPI-anchored proteins, it is possible that its overexpression leads to imbalance of transport of these proteins involved in chitin synthesis or cell wall maintenance.
Examples of such proteins are Gas1p, Crh1p, and Crh2p (Table 4.3). Cells bearing deletions of CRH1 and CRH2 were shown to be sensitive to Calcofluor
White and Congo Red, which is also a dye that interfere with cell wall assembly
(Rodriguez-Peña, 2000). Since the concentration of Calcofluor White used in this study was relatively high, it is probable that the level of Crh1p and Crh2p was not high enough to cope for this surge of the dye. However, more experiments with
94 Calcofluor White on p24 co-overexpression are desirable to obtain a better understanding of the effect of this antifungal dye on the cells.
Another phenotype worth investigating is the Kar2p secretion caused by
UPR activation. Strains bearing a deletion of some p24 genes lead to the secretion of this ER chaperone into the medium (Schimmoller, 1995; Belden, 2001a). It would be interesting to test the effect of p24 co-overexpression on the UPR.
4.4 Expression of recombinant p24 proteins
In order to validate some of the interactions detected by MYTHS, I expressed several p24 proteins in insect Sf9 cells for in vitro studies. TMP21L-
His6 was the only p24 that was soluble, but the yield after purification was extremely low and not detected by Coomassie Blue staining. I then considered coexpression in bacteria since a p24 protein might needs its interactors to be stable. I attempted coexpression for several pairs of p24s based on MYTHS results. Again, only His6-TMP21L was expressed abundantly and soluble, but further experiments revealed that TMP21L did not need its partner p24AL to be stable and soluble. This is in agreement with another study that has shown that p24s proteins were mostly in monomers or dimers (Jenne, 2002). However, primary characterization experiments have shown that TMP21L was unstructured.
A solution to this issue is to purify His6-TMP21L in denaturing conditions followed by refolding assays.
Conclusions
In this study, I have shown by MYTHS that pairwise interactions, among the eight p24 proteins of S.cerevisiae are specific and mostly occur through their
95 GOLD domain with contribution from the DOG sequence for some pairs. Only two human p24 proteins (TMP21 and p24A) interact, also through their GOLD domain. However, none of the interactions could be proven physically in vitro as the p24 proteins were not expressed or insoluble except for TMP21, which could be purified in high yield, but was unstructured. Mutagenesis experiments suggest that Erp1G and Erp6G interact differently with Erp4G and this strategy could be used to define the interactions between other p24 proteins. Co-overexpression of p24s in yeast cells lead to growth defect and large cell phenotype for a subset of combinations. Microscopy work showed that the antifungal dye Calcofluor White has an effect on cells double overexpressing ERV25 and probably others as well.
Together, these experiments, combined with the data in the literature, revealed that the p24 proteins might associate with each other for transport of
GPI-anchored proteins involved in cell wall maintenance or cell cycle pathways.
96 APPENDIX A
List of Primers
Table A1. Cloning ERp6 DOG into pDONR221 entry vector Primers Sequence (5’ 3’) ERp6D-F GGGGACAAGTTTGTACAAAAAAGCAGGCTCAGGTTTT GAAGCAATGTTGGACATGCAAAGAAAAGAAACC ERp6D-R GGGACCACTTTGTACAAGAAAGCTGGGTTGCGAGAAT TCACAGACTCTGATATGTCTC
Table A2. PCR Random Mutagenesis of ERp4 GOLD Primers Sequence (5’ 3’) ERp4G-F CTGAGGAATTAATATTTTAGCACTTTGAAAAGTCGACA TGCGCGTTTTTACTTTGATTGCGATTTTGTTTAGTTC ERp4G-R CACCCTCGAAAAGGAAAAGGAAATTGTGTCGACAATG GATGAAAAGAACCAAAATTCTTTGCTACTG
Table A3. Amplification of HygMX and KanMX Cassettes Primers Sequence (5’ 3’) MX4-F ACATGGAGGCCCAGAATACCC MX4-R CAGTATAGCGACCAGCATTCAC
Table A4 Primers to verify the ERp4 Knockout Primers Sequence (5’ 3’) ERp4prom-F GGCGCGCCGCTAGCGGGATATGTTCTCCT ERp4term-R CTCGAGCATCATTTGAGCATACTTCAATCG
Table A5. Cloning into p425, p426 and pADH426 plasmids Primers Sequence (5’ 3’) ERp3-BamHI-F TTTTGGATCCATGTCCAATTTATGTGTACTTTTC ERp3-XhoI-R TTTTCTCGAGTTACACATTATGCTTTCTCGATTC ERp4-BamHI-F TTTTGGATCCATGCGCGTTTTTACTTTGATTGCG ERp4-XhoI-R TTTTCTCGAGCTATACGTAGTTTTTTTGGCGACT ERp5-BamHI-F TTTTGGATCCATGAAATATAATATAGTGCATGGA ERp5-XhoI-R TTTTCTCGAGTTATACTACCTTCTGTTTGACAAA ERp6-BamHI-F TTTTGGATCCATGTTATCACACTACATCTTCCTG ERp6-XhoI-R TTTTCTCGAGTCATAATACTTTCTGCTTAACAAA TMED2-BamHI-F TTTTGGATCCATGGTGACGCTTGCTGAACTG TMED2-XhoI-R TTTTCTCGAGTTAAACAACTCTCCGGACTTC TMED10-BamHI-F TTTTGGATCCATGTCTGGTTTGTCTGGCCCA TMED10-XhoI-R TTTTCTCGAGTTACTCAATCAATTTCTTGGC
97 Table A6. Cloning into pFASTBac11-Cpo-His6 plasmid Primers Sequence (5’ 3’) TMP21L-RsrII-F AACGGTCCGATCTCCTTCCATCTGCCCATT TMP21L-RsrII-R AACGGACCGCCCCGAGTGTTTGTTGACT p24AL-RsrII-F AACGGTCCGTATTTCGTTAGCATCGACGCC p24AL-RsrII-R AACGGACCGCCTCTGCTGTTTGTGTTGTC ERp1-RsrII-F AACGGTCCGCAAATTTACGATGACCAATTACAA AATTACAGAGA ERp1G-RsrII-R AACGGACCGCCCACTTGGAATTCAACGTCA ERp1L-RsrII-R AACGGACCGCCACGAGAATTAACAGCTTCAGAG ERp4-RsrII-F AACGGTCCG TTGACAGGTGGGAATTTCGAG ERp4G-RsrII-R AACGGACCGCCCTTTTCGAGGGTGATTTC ERp4L-RsrII-R AACGGACCGCCTCTTGACTCAGTAGAACTCACA ERp6-RsrII-F AACGGTCCG CCCAGTTATAATGACTATGGCAT ERp6G-RsrII-R AACGGACCGCCGACTTCAAATTCGATTTCTAAC ERp6L-RsrII-R AACGGACCGCCGCGAGAATTCACAGACTCTG
Table A7. Cloning into pETDuet modified plasmid Primers Sequence (5’ 3’) TMP21L-BamHI-1F TTTTGGATCCATCTCCTTCCATCTGCCCATT TMP21L-NotI-1R TTTTGCGGCCGCCTACCGAGTGTTTGTTGACTC GTT p24AL-BamHI-1F TTTTGGATCCTATTTCGTTAGCATCGACGCC p24AL-NotI-1R TTTTGCGGCCGCCTATCTGCTGTTTGTGTTGTC GTT
Table A8. Primers used for sequencing Primers Sequence (5’ 3’) pFB11-Cpo-His6-F CTACTAGTAAATCAGTCACACCAA pFB11-Cpo-His6-F GTGTGGGAGGTTTTTTAAAGCAA pRSFDuet-1F CGGGATCTCGACGCTCTCCCTTATGCGACT pRSFDuet-1R ATGTATATCTCCTTCTTATACTTAACTAAT pRSFDuet-2F GTCGAACAGAAAGTAATCGTATTGTACACG pRSFDuet-2R GGTTTATTGACTACCGGAAGCAGTGTGACC pETDuet-1F CGGCGTAGAGGATCGAGA pETDuet-1R TCTCCTTCTTATACTTAACTAATA pETDuet-2F GGCCGCATAATCGAAATTAAT pETDuet-2R GCCAATCCGGATATAGTTCCT
98 Table A9. Cloning into pRSFDuet plasmid Primers Sequence (5’ 3’) TMP21L-SalI-1F TTAAGTCGACATCTCCTTCCATCTGCCCATT TMP21L-NotI-1R TTAAGCGGCCGCCTACCGAGTGTTTGTTGACTCGT TGGT p24AL-EcoRV-2F TTAAGATATCATGTATTTCGTTAGCATCGACGCCC AT p24AL-XhoI-2R TTAACTCGAGTCTGCTGTTTGTGTTGTCGTTGAT p24AL-SalI-1F TTTTGTCGACTATTTCGTTAGCATCGACGCC p24AL-NotI-1R TTTTGCGGCCGCCTATCTGCTGTTTGTGTTGTC GTT TMP21L-EcoRV-2F TTTTGATATCATGATCTCCTTCCATCTGCCCATT TMP21L-XhoI-2R TTTTCTCGAGCCGAGTGTTTGTTGACTCGTT ERp1G- SalI-1F TTAAGTCGACCAAATTTACGATGACCAATTACAA ERp1G--NotI-1R TTAAGCGGCCGCCTACACTTGGAATTCAACGTCA AT ERp4G-BglII-2F TTAAAGATCTATGTTGACAGGTGGGAATTTCGAG ERp4R-XhoI-2R TTAACTCGAGCTTTTCGAGGGTGATTTCAAC ERp4G- SalI-1F TTAAGTCGACTTGACAGGTGGGAATTTCGAG ERp4G-NotI-1R TTAAGCGGCCGCCTACTTTTCGAGGGTGATTTCA AC ERp6G-BamHI-2F TTAAGGATCCATGCCCAGTTATAATGACTATGGC
ERp6G-XhoI-2R TTAACTCGAGGACTTCAAATTCGATTTCTAA ERp3G- SalI-1F TTAAGTCGACCAGCAAGGAGAGAGCAATGACTTT ERp3G--NotI-1R TTAAGCGGCCGCCTATTGGCGTTCACAATTGTAC TT ERp5G-EcoRV-2F TTAAGATATCATGAAGGATGGTCTGTTTGAAGAG ERp5G-XhoI-2R TTAACTCGAGAACATTGCCAATTTCTAGTTC EMP24- SalI-1F TTAAGTCGACCAATCCAGTAGCCAGCTGACT EMP24--NotI-1R TTAAGCGGCCGCCTATACCACCCCATGAATGTTG AA ERV25G-BglII-2F TTAAAGATCTATGTCTGTTGGTGATGGACAGAAA ERV25G-XhoI-2R TTAACTCGAGGATCTTGTTCCAGTCACGAGC
99 APPENDIX B
Table B1. List of Plasmids for yeast sequences Insert Available in plasmids ERp1 pDONR221, pMYTHS5-SS, pMYTHS6-SS, p425, p426 ERp2 pDONR221, pMYTHS5-SS, pMYTHS6-SS, p425, p426 ERp3 pDONR221, pMYTHS5-SS, pMYTHS6-SS, p425, p426 ERp4 pDONR221, pMYTHS5-SS, pMYTHS6-SS, p425, p426, pADH426 ERp5 pDONR221, pMYTHS5-SS, pMYTHS6-SS, p425, p426 ERp6 pDONR221, pMYTHS5-SS, pMYTHS6-SS, p425, p426 EMP24 pDONR221, pMYTHS5-SS, pMYTHS6-SS, p425, p426 ERV25 pDONR221, pMYTHS5-SS, pMYTHS6-SS, p425, p426 ERp1 Luminal pDONR221, pMYTHS3-SS, pMYTHS4-SS, pFASTBac-Cpo-His6 ERp2 Luminal pDONR221, pMYTHS3-SS, pMYTHS4-SS ERp3 Luminal pDONR221, pMYTHS3-SS, pMYTHS4-SS ERp4 Luminal pDONR221, pMYTHS3-SS, pMYTHS4-SS, pFASTBac-Cpo-His6 ERp5 Luminal pDONR221, pMYTHS3-SS, pMYTHS4-SS ERp6 Luminal pDONR221, pMYTHS3-SS, pMYTHS4-SS, pFASTBac-Cpo-His6 EMP24 pDONR221, pMYTHS3-SS, pMYTHS4-SS Luminal ERV25 pDONR221, pMYTHS3-SS, pMYTHS4-SS Luminal ERp1 GOLD pDONR221, pMYTHS3-SS, pMYTHS4-SS, pFASTBac-Cpo-His6, pRSFDuet ERp2 GOLD pDONR221, pMYTHS3-SS, pMYTHS4-SS ERp3 GOLD pDONR221, pMYTHS3-SS, pMYTHS4-SS ERp4 GOLD pDONR221, pMYTHS3-SS, pMYTHS4-SS, pFASTBac-Cpo-His6, pRSFDuet ERp5 GOLD pDONR221, pMYTHS3-SS, pMYTHS4-SS ERp6 GOLD pDONR221, pMYTHS3-SS, pMYTHS4-SS, pFASTBac-Cpo-His6, pRSFDuet EMP24 GOLD pDONR221, pMYTHS3-SS, pMYTHS4-SS ERV25 GOLD pDONR221, pMYTHS3-SS, pMYTHS4-SS ERp1 DOG pDONR221, pMYTHS3+SS, pMYTHS4+SS ERp2 DOG pDONR221, pMYTHS3+SS, pMYTHS4+SS ERp3 DOG pDONR221, pMYTHS3+SS, pMYTHS4+SS ERp4 DOG pDONR221, pMYTHS3+SS, pMYTHS4+SS ERp5 DOG pDONR221, pMYTHS3+SS, pMYTHS4+SS ERp6 DOG pDONR221, pMYTHS3+SS, pMYTHS4+SS EMP24 DOG pDONR221, pMYTHS3+SS, pMYTHS4+SS ERV25 DOG pDONR221, pMYTHS3+SS, pMYTHS4+SS
100 Table B2. List of Plasmids for human sequences Insert Available in plasmids TMED1 Luminal pDONR221, pMYTHS3-SS, pMYTHS4-SS TMED2 Luminal pDONR221, pMYTHS3-SS, pMYTHS4-SS, pFASTBac-Cpo-His6, pRSFDuet-I, pETDuet-I modified TMED3 Luminal pDONR221, pMYTHS3-SS, pMYTHS4-SS TMED4 Luminal pDONR221, pMYTHS3-SS, pMYTHS4-SS TMED5 Luminal pDONR221, pMYTHS3-SS, pMYTHS4-SS TMED7 Luminal pDONR221, pMYTHS3-SS, pMYTHS4-SS TMED9 Luminal pDONR221, pMYTHS3-SS, pMYTHS4-SS TMED10 Luminal pDONR221, pMYTHS3-SS, pMYTHS4-SS, pFASTBac-Cpo-His6, pRSFDuet-I, pETDuet-I modified TMED2 GOLD pDONR221, pMYTHS3-SS, pMYTHS4-SS TMED10 GOLD pDONR221, pMYTHS3-SS, pMYTHS4-SS APP pDONR221, pMYTHS5-SS, pMYTHS6-SS APPΔTMD pDONR221, pMYTHS3-SS, pMYTHS4-SS APH-1 pDONR221, pMYTHS5-SS, pMYTHS6-SS PEN-2 pDONR221, pMYTHS3-SS, pMYTHS4-SS PSEN1 pDONR221, pMYTHS3+SS, pMYTHS4+SS PSEN2 pDONR221, pMYTHS3+SS, pMYTHS4+SS NCSTN pDONR221, pMYTHS5-SS, pMYTHS6-SS
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