Eukaryotic Oligosaccharyltransferase - A structure-function characterisation of Saccharomyces cerevisiae Stt3 protein

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1 Research Collection Doctoral Thesis Eukaryotic Oligosaccharyltransferase - A structure-function characterisation of Saccharomyces cerevisiae Stt3 protein Author(s): Ngwa, Elsy Mankah Publication Date: 2017 Permanent Link: Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library

2 DISS. ETH NO Eukaryotic Oligosaccharyltransferase- A structure-function characterisation of Saccharomyces cerevisiae Stt3 protein A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH ZURICH (Dr. sc. ETH Zurich) presented by Elsy Mankah Ngwa MPH in Public Health, Nutrition and Physical Activity, University of Westminster, United Kingdom MSc. In Microbiology, University of Buea, Cameroon born on citizen of Bafut Mezam, Cameroon accepted on the recommendation of Prof. Dr. Markus Aebi Prof. Dr Kaspar Locher Pro. Dr. Yves Barral Prof. Dr Reid Gilmore 2017

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4 Acknowledgements First and foremost, I would like to express my profound gratitude to my supervisor Prof. Markus Aebi for having given me the opportunity to do my PhD in his group. This has been a remarkable journey and an important chapter in my life. I am grateful for his continuous support, for his patience, motivation and immense knowledge. Not only has he taught me how to do good science, his enthusiasm and love of history and evolution has been inspiring and has greatly widen my perspective on life. I am truly thankful to my thesis committee: Prof. Kaspar Locher, Prof. Yves Barral and Prof. Reid Gilmore, for their insightful comments and fruitful discussions during our thesis committee meetings. I owe gratitude to the staff at the functional genomics centre especially Dr. Nathalie Selevsek and Dr. Asa Wahlander for their support and assistance with mass spectrometric measurements, for keeping the machines running and for answering all my questions. A big thanks goes all the former Aebians who made the beginning of this journey tolerable and enjoyable. Special thanks to Jörg for introducing me to the world of yeast genetics, Susanne and Robert for interesting and fruitful scientific discussions and collaboration, Steffie and David for being there during the difficult times. I would also like to thank current Aebians for creating a conducive and fun working environment and for the many scientific and non-scientific exchanges. In particular, I want to thank Marie-Estelle, Jillian, Kristina, Tim and my lunch buddies Chia-Wei, Christina, Anja and Martina. I am profoundly thankful to Nana and Robinson for their academic input and for being there. Special thanks also goes to all members of the African Students Association of Zurich for providing an African cultural environment. It was an honor to be part of this group, to share the rich diversity of culture, food, music and dance styles from our continent. A very big 'MIYAH' to my mum, grandma, younger sisters, niece and nephews, who have always supported and encourage me even from a distance. Hugs to my wonderful friends Vivian, Daniel, Humphrey, Clotilde, Valerie and Mefor for the moral support over the years.

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6 Table of Contents Thesis summary... i Résumé de thèse... iii Chapter 1:... 1 Introduction Chapter 2: Insights into the function(s) of Saccharomyces cerevisiae Stt3 protein: A structurefunction characterization Chapter 2: Supplementary information Chapter 2B: Further characterisation of S cerevisiae Stt3p Chapter 3: Conclusions and future perspectives Chapter Appendix Chapter 5... Error! Bookmark not defined. Curriculum vitae

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8 Thesis summary N-linked glycosylation is the most common post translational modification of proteins in archaea, bacteria and eukaryotes that influences protein folding, stability, solubility and function. In eukaryotes, oligosaccharyltransferase (OST) transfers preassembled glycan chains from a lipid-linked oligosaccharide donor (Dol-PP-GlcNAc2 Man9 Glc3) onto asparagines within the consensus sequon N-X-S/T (where X is any amino acid but proline) on nascent polypeptides in the endoplasmic reticulum (ER) lumen. Chapter one gives an overview of N-linked glycosylation pathway and its biological relevance. It describes the biosynthesis of the building blocks needed for the assembly of the oligosaccharide substrate. The second part of chapter one discusses the central enzyme of the N-glycosylation pathway, OST, outlining its subunit composition, function of the subunits, assembly of the subunits into the complex for higher eukaryotes and the mechanism of the reaction. Furthermore, an overview of the different analytical tools and methods used for the characterisation and elucidation of OST function are presented. The last part of this chapter discusses the importance of N-linked glycosylation in protein folding and ER quality control for the elimination of misfolded and unassembled proteins from the ER. Stt3p is a polytopic membrane proteins that is highly conserved in all domains of life. Stt3p catalyses the N-glycosylation reaction as a single transmembrane protein in prokaryotes and lower eukaryotes and in higher eukaryotes it functions as part of a multi-subunit protein complex. The aim of this project is to characterize Stt3p in S. cerevisiae with respect to the role it plays in N-linked glycosylation of proteins and formation of the OST complex in vivo. Chapter two reports a reverse genetics approach for generating and analysing Stt3p point-mutants selected based on its postulated structure. It further outlines the quantitative mass spectrometric approaches used for the analysis of OST complex assembly and N-glycosylation-site occupancy of yeast proteins in Stt3p mutants that influence OST activity. Chapter 2B addresses the function of the luminal domain of the Stt3 protein and other mutations of highly conserved amino acid residues. The final chapter summarises the main findings of this thesis and suggest an outlook for future research. In brief, we identified amino acid residues involved in the catalytic centre, complex assembly, or which affects OST activity. i

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10 Résumé de thèse La N-glycosylation est la modification post-traductionnelle des protéines la plus commune chez les archaea, les bactéries et les eucaryotes, elle influence mise en conformation, la stabilité, la solubilité et les fonctions des protéines. Chez les eucaryotes, l'oligosaccharyltransférase (OST) transfert à partir d'un donneur oligosaccharidique lie à un lipide (Dol-PP-GlcNAc2 Man9 Glc3), une chaine glycanique sur l'asparagine du séquon N-X-S/T (où X est n'importe quel acide aminé sauf la proline) d'une chaine polypeptidique naissante dans l'espace luminal du réticulum endoplasmique (RE). Le chapitre 1 présente une vue d'ensemble de la voie de N-glycosylation et sa relevance biologique. Il décrit la biosynthèse des composants nécessaire pour l'assemblage du substrat oligosaccharidique. Dans un second temps, il décris l'enzyme centrale de la voie de N-glycosylation, l'ost, en soulignant la composition de ses sous-unités, leurs fonctions, leur assemblage en complexes chez les eucaryotes supérieurs et son mécanisme de réaction. De plus, il y est présenté une description des différents outils analytiques et des méthodes utilisées pour la caractérisation et l'élucidation des fonctions de l'ost. Finalement, il présente l'importance de la N-glycosylation dans la mise en conformation protéique et le contrôle qualité, afin d'éliminer du réticulum les protéines mal conformées ou non-assemblées. Stt3p est une protéine de type II qui est fortement conservée à travers tous les domaines de la vie. Stt3p catalyse la réaction de N-glycosylation, elle est constituée d'une protéine transmembranaire unique chez les procaryotes et eucaryotes inférieurs, chez les eucaryotes supérieurs elle est un élément d'un complexes multi-protéique. Le but de ce projet est de caractériser Stt3p chez S. cerevisiae en ce qui concerne son rôle dans la N-glycosylation des protéines et la formation du complexe de l'ost in vivo. Le chapitre 2 explique l'approche de génétique inverse utilisée pour générer et analyser les mutants ponctuels de Stt3p sélectionnés sur la base de sa structure tridimensionnelle. Il souligne, par ailleurs, les techniques quantitatives de spectrométrie de masse utilisées pour l'analyse de l'assemblage du complexe OST, ainsi que l'occupation des sites de N-glycosylation des protéines de levure, qui influencent l'activité de l'ost chez les cellules mutées dans Stt3p. Le chapitre 2B propose les fonctions potentielles du domaine luminal et d'autres mutations d'acides aminés fortement conserves de la protéine Stt3p. iii

11 Le Chapitre final résume les principales conclusions de cette thèse, dans laquelle nous avons identifié les acides aminés impliqués dans le centre catalytique, l'assemblage du complexe, ou qui affectent l'activité de l'ost, il suggère également les futures directions de recherche iv

12 Chapter 1: Introduction 1

13 1.1 N-linked glycosylation: Biological relevance Post-translational modifications are a set of protein-processing events and principal regulators of protein function, activity, localization and interaction. N-linked glycosylation is the most frequent of such modifications and involves the attachment of a carbohydrate moiety onto secretory or membrane-bound proteins of eukaryotes and a few prokaryotes 1. This evolutionary conserved and complex process which is essential for cell viability in eukaryotes occurs in two main steps; the assembly of the oligosaccharide donor substrate and the transfer of assembled substrate onto newly synthesised proteins. Oligosaccharyltransferase (OST), the key enzyme of this pathway catalyses the transfer of the glycan to selected asparagine (N) residues of the consensus sequon N-X-S/T (where X is any amino acid except proline) of polypeptides being translocated into the lumen of the endoplasmic reticulum (ER) 2. Upon transfer, the glycoproteins traverse the Golgi apparatus where their glycans are modified by a multitude of hydrolases and glycosyltransferases. Golgi modifications generate a vast diversity of glycoproteins which span the plasma membrane or are secreted from the cell 3,4. N-linked glycans can influence structure, stability and solubility of proteins in the ER, Golgi and on the cell surfaces. In addition, they play a vital role in the folding and transport of proteins acting as universal tags which determine when a protein can be refolded, transported to the Golgi or targeted for degradation 5. On fully-folded glycoproteins, glycans are involved in a multitude of functions such as, changing the properties of secreted proteins and hormones, participating in cell-to-cell interaction by determining the specificity for membrane receptors in innate immunity, or by acting as targets for many pathogens and toxins 4,6 8. Because N-glycosylation is involved in several cellular processes, defects in the assembly pathway and/or modification of glycans affect developmental processes in humans. These human genetic diseases are collectively called Congenital Disorders of Glycosylation (CDGs). Mutations in genes affecting the biosynthesis of the lipid-linked oligosaccharide(llo) precursor and transfer of the glycan to substrate proteins in the ER are classified as Type I CDGs 9, while those affecting trimming and processing of oligosaccharide after they are transferred to proteins are known as Type II CDGs 10. Type I CDGs result in hypoglycosylation of proteins affecting many organs of the body and primarily delay 2

14 the development of the brain causing mental disabilities. Mutations in the human OST subunits DDOST, TUSC3, MAGT1, STT3A and STT3B are known to cause CDGs with severe mental retardation More than 70% of all human proteins are N- glycosylated 16 and a complete loss of glycosylation is lethal in all eukaryotes, emphasizing the importance of N-linked glycosylation. 1.2 Assembly of oligosaccharide substrate The oligosaccharide in higher eukaryotes composed of Glc3Man9GlcNAc2 is preassembled on a dolichol lipid carrier in a process that occurs on both sides of the ER membrane. LLO biosynthesis is carried out by a series of membrane-bound glycosyltransferases, which are encoded by asparagine-linked glycosylation (ALG) genes (Fig. 1). In the cytoplasmic phase, ALG gene products use nucleotide activated sugars such as UDP N-acetylglucosamine (UDP-GlcNAc) and GDP-Mannose (GDP- Man) to assemble a Man5GlcNAc2 oligosaccharide on the isoprenoid lipid, dolichol. This processes begins with the addition of an N-acetylglucosamine phosphate residue to dolichol phosphate by the ALG 7 gene product N-acetylglycosamyl phosphate transferase (GPT). GPT is sensitive to the drug tunicamycin, as such has been used as a tool to study the effects of blocking N-glycosylation on cellular processes like protein folding 17,18. Addition of the second GlcNAc residues is catalysed by the protein complex encoded by ALG13 and ALG The ALG1 gene product β-1, 4-mannosyltransferase adds the first mannose residue on to Dol-PP-GlcNAc2 20. Alg1 mutants have a temperature sensitive phenotype and defects in the human homologue halg1 are known to cause the CDG termed CDG-Ik 19. The addition of the second and third mannoses are catalysed by ALG2 generating the substrate for ALG 11 which adds the final two mannoses 21,22. Man5GlcNAc2, which is the final product of biosynthesis on the cytosolic phase is eventually flipped into the ER Lumen by Rft1p 23 where luminal glycosyltransferases AlG3, ALG9, ALG12, ALG6, ALG8 AND ALG10 use dolichol-phospho-sugars such as Dol-P-Man and Dol-P-Glc to complete the assembly of the Glc3Man9GlcNAc2 for transfer 23 (Fig. 1). Luminal-oriented glycosyltransferase unlike the cytosolic ones are not essential for cell viability and their deletion only results in hypoglycosylation of proteins. Addition of terminal α-1, 2-linked glucose residue by ALG10 act as signal that 3

15 the oligosaccharide is complete and ready to be transferred 24. Such recognition specificity ensures that completely assembled glycans are primarily transferred to proteins. Figure1: Asparagine-linked glycosylation pathway in S. cerevisiae. The LLO precursor (Glc3Man9GlcNAc2) for N-glycosylation is synthesized in a process that occurs on both sides of the ER membrane catalysed by conserved glycosyltransferases encoded by ALG genes. The mature LLO on the luminal side of the ER is transferred en bloc onto substrate proteins by the oligosaccharyltransferase. Modified from Breitling and Aebi, Oligosaccharyltransferase Subunit composition Oligosaccharyltransferase is the key enzyme in the N-linked glycosylation pathway and catalyses the transfer of LLOs to newly synthesized polypeptides emerging from the translocon. Evolutionarily, OST activity is conserved in all domains of life, but organisms possess OSTs with varying degrees of structural complexity. It is expressed as a multi-protein complex with additional proteins interacting with the core Stt3p in higher eukaryotes. For example, the OST complex of our model organism S. cerevisiae 4

16 has eight subunit-proteins while the human parasites Plasmodium falciparum and Cryptosporidium parvum express complexes of four (Ost1p, Ost2p, Wbp1p and Stt3p) and six (Ost1p, Ost2p, Wbp1p, Stt3p, Ost4p, Ost3p or Ost6p) subunits respectively 25. OST activity is most studied in the yeast S. cerevisiae, the complex of which is composed of eight subunits encoded by OST3, OST4, OST5, OST6 and the essential genes OST1, OST2, WBP1, SWP1 and STT3. These complexes are expressed in two isoforms incorporating either the Ost3 or Ost6 proteins 26,27. The mammalian OST complex encodes subunits homologous to those found in yeast (yeast homologues in brackets) N33/Tusc3 and IAP3 (OST3 and OST6), ribophorin I (OST1), DAD1 (OST2), OST48 (WBP1), ribophorin II (SWP1), OST4 (OST4) and STT3A and STT3B (STT3). STT3A and STT3B are incorporated into distinct isoforms which show different activity profiles, and have the ability to discriminate between donor substrates with different oligosaccharide structures 28. In contrast, the OST of lower eukaryotes like the kinetoplastids is a single-subunit enzyme consisting of only STT3 that is self- sufficient for glycosylation. Archaea and eubacteria OSTs are also single-subunit enzymes, consisting of the AglB and PglB respectively (homologues of eukaryotic STT3) 25,29. Intriguingly, a gene duplication event in some Kinetoplastids allow them to encode several paralogues of Stt3p which are capable of functionally replacing the complex OSTs as single proteins 30,31. The human parasites Leishmania major for example encodes four paralogues (LmSTT3A, LmSTT3B, LmSTT3C and LmSTT3D) while Trypanosoma brucei encodes three (TbSTT3A, TbSTT3B and TbSTT3C). When expressed in yeast cells, these paralogues glycosylate yeast N-X-S/T sites and are therefore able to keep the cells viable in the absence of a functional endogenous OST. For this reason, kinetoplastid paralogues of Leishmania major (LmSTT3D) or Trypanosoma brucei (TbSTT3C) are very important as a research tool in yeast genetics, and are often used to complement the deletion of the endogenous protein when studying potentially lethal mutations. OST has therefore evolved with strategies that broaden or diversify its substrate range and glycosylation ability, in order to generate the different complex glycoproteomes observed in nature. It has done so by either adding more subunits onto the catalytic Stt3p core in complex- forming OSTs, or by duplicating the Stt3 protein in lower eukaryotes. 5

17 Structure and functions of OST subunits Considering the importance of the OST in protein glycosylation, researchers have in the past few decades cloned and expressed all the subunits of the multimeric OST and the main challenge remains to understand their functions and how they interact with each other. A combination of yeast genetics and biochemistry experiments led to the proposition that the eight subunits of the yeast OST are organized in three putative sub-complexes; Ost1-Ost5, Swp1-Wbp1-Ost2 and Stt3-Ost3-Ost4 25,26. Crystal and NMR structures have been obtained for Ost4p 32, Ost6 33, soluble domains of Stt3p from archaea 34 and yeast 35, full length PglB of bacteria 36, full length AglB from Archaea 37 and a 12-Å resolution of the whole complex 38. These structures have shed some light on the functions of some of the subunits but questions about the mechanism of catalysis at least for eukaryotic OSTs still remain unanswered. Experimental data from studies with yeast and mammalian OSTs together with the identification of bacterial OSTs consisting of the single protein PglB have led to the conclusion that STT3 encodes the catalytically active OST subunit 1,26,29,39. STT3A isoform of the plant Arabidopsis is thought to directly or indirectly play an important role in growth and development of the plant by controlling adaptive responses to salt/osmotic stress 40. Though not fully experimentally proven, other subunits are thought to fine-tune and regulate the N-linked glycosylation process. The crystal structure of PglB, the bacteria homologue of the catalytic STT3 subunit of yeast has provided the molecular basis for discerning the N-linked glycosylation reaction mechanism 36. The mechanism proposes a three-stage catalytic cycle where the peptide substrate binds to the enzyme and engages the flexible external loop 5 (EL5) which restricts the movement of the peptide, followed by the transfer of the LLO substrate onto the bound peptide. In the final stage, the EL5 disengages from the catalytic site causing the release of the glycosylated peptide (Fig. 2). Regardless of the fact that several differences exist in the N-linked glycosylation process, the general mechanism is conserved and we can tentatively assume that the mechanism proposed for the bacterial OST will be similar in eukaryotes, this remains to be proven though. 6

18 Figure 2: Mechanism of N-linked glycosylation of bacterial OST. Crystal structure of PglB showing different stages in the catalytic reaction. (1) glycosylation sequon binding with engagement of EL5; (2) LLO binding followed by glycosylation; (3) disengagement of EL5, release of glycosylated sequon and lipid-pyrophosphate (PP) 36. Ost3/Ost6p are suggested to have oxidoreductase activity and help with efficient glycosylation of translocated polypeptides by directly affecting their oxidative folding. Ost3/Ost6p transiently binds to substrate peptides through mixed disulphide bond formation that slows down rapid folding of peptides, and by so doing provides unfolded peptides for more efficient glycosylation of sequons close-by 33,41,42. It is also known that complexes that incorporate the different isoforms; Ost3p or ost6p, differentially glycosylate protein sites with Ost3p complexes having a preference for hydrophobic and polar peptides while Ost6p complexes bind to hydrophobic and acidic peptides 33. The functions of all the other subunits are less known. Ost4p is a short transmembrane protein which is essential for cell viability at 37 C but not at 25 C, and its deletion impairs OST activity through destabilisation of the complex OST5, though not essential for vegetative growth, is required for maximal OST activity 47. These two subunits are therefore presumed to play a structural role within the complex. OST1 7

19 activity is required for vegetative growth of cells and its deletion results in complex destabilisation. Ribophorin 1, the mammalian homologue of yeast OST1 has been shown to regulate acceptor peptide delivery to the OST 48,49. Genomic disruptions of the mammalian homologues; OST48 (Wbp1p), ribophorin II (Swp1) and DAD1 (Ost2p) destabilise OST complex causing lethality, however, functions other than a structural role are not yet defined for these subunits. DAD1 has been shown to be involved in apoptosis, though, no similar function has been described for its yeast counterpart OST complex assembly OST is a large multimeric complex composed of eight subunit proteins. How these subunit proteins assemble into a complex is vital for the overall functioning and regulation of the complex. It is a well-known phenomenon that protein-complex assembly occurs in an ordered manner passing through the formation of intermediates with subunits interacting in a time sequence and not randomly 50. Although assembly progresses via stable sub-complexes, not all proteins interacting in the final complex form stable intermediates, and not all stable intermediates are active. Assembly is therefore tightly regulated with the aid of chaperons to avoid the accumulation of aggregated proteins, or incomplete intermediates that might be toxic or might sequester substrate and compete with the complete complex thereby affecting overall function 50. To further avoid such detrimental consequences, activity sometimes is only conferred by the complete final complex. In 2015, Mueller and colleagues 46 introduced different perturbations to the yeast OST complex like overexpression or deletion of subunits and used a sensitive SILAC-SRM method to evaluate complex assembly. They could show that the basic principle of membrane protein complex assembly mentioned above also applies to the yeast OST complex. Through their work and those of others, it is now suggested that during complex assembly, Wbp1p, Swp1p, and Ost2p form an intermediate sub-complex, while Ost1p, Ost5p, and Stt3p form another. After the formation of these two intermediates, Ost4p enters the complex and anchors Ost3p or Ost6p to form the final active complex 26,45,46. Defects in this assembly process affect enzyme activity which might lead to accumulation of unfolded proteins in the cell, with detrimental effects if not corrected through refolding or degradation by the ER quality control system. 8

20 1.3.4 Analysis of OST function This section discusses the analytical tools and methods that have been used to characterise protein N-linked glycosylation, and to elucidate the functions of OST and its subunits Biochemical and molecular biological analysis of OST activity Protein separation approaches such as SDS PAGE and 2D electrophoresis followed by blotting with specific antibodies or lectins are commonly used to characterize glycosylation patterns in cells, thus, OST activity. Carboxypeptidase Y (CPY), a vacuolar protein harbouring four glycosylation sites is the model protein used to analyse OST activity in yeast. In a hypoglycosylation state, different glycoforms of CPY can be visualized on a gel owing to the differences in their molecular masses 23. Other methods such as Cross-linking and co-immunoprecipitation techniques are also commonly used and have been instrumental in the identification of WWDYG motif on Stt3p which serves as the peptide substrate binding site 51,52. Over the years, a combination of different chromatographic and immunoaffinity techniques have been used to purify the OST complex from different yeast and mammalian sources for in vitro activity studies 53,54. The most common in vitro activity assays used either biosynthetically or chemo-enzymatically synthesised LLOs as donor substrate and lectin binding mechanism for separation. For example, in 2002, Srinivasan and Coward used biotinylated peptides as acceptor substrate and radiolabelled chitobiose as donor substrate to monitor OST reaction. These assays were based on lectin binding separation and quantification with HPLC chromatography analysis and scintillation counting 55. Such experiments yielded rapid results with high reproducibility. However, they were tedious, potentially hazardous and very time consuming. More so, the structure of the LLO or at least the N-glycan structure had to be known in order to design proper assays. Nowadays, the preferred in vitro assays are based on mobility separation on SDS-PAGE and detection by fluorescence gel imaging of a synthetic peptide that has a fluorophore attached to it. This method is highly sensitive and the most commonly used fluorophores; carboxytetramethylrhodamine (TAMRA) and Carboxyfluorecein (CF) are easy to quantify and are stable over the long- 9

21 term In addition, no previous knowledge of LLO or glycan structure is required in such assays, which are also radioisotope-independent Genetics In the past decades, genetic analyses have been done in bacteria, yeast and mammalian systems, but yeast (S. cerevisiae), remains the best model eukaryotic organism for biological studies. The ease of genetic manipulation of yeast makes it handy for analysing and functionally characterising genes from other organisms 59. Yeast cells are stable in the haploid and diploid stages and are easily transformable with a wide range of plasmids. Yeast genes that complement a specific auxotrophy allow the selection of plasmids that have been transformed. These properties have facilitated the isolation and generation of OST mutants, using, for example, mating and complementation assays, tetrad analysis, gene mapping and replica plating 60. Such mutants have been used to understand the N-linked glycosylation pathway and structure-functional relationships of the different components of the OST. Genes involved in the biosynthesis of the LLO substrate of the OST have been isolated in forward genetic screens and their characterization have provided insights to the function of the pathway 19,61,62. wbp1-2, a temperature sensitive allele of Wbp1p with reduced OST activity was isolated in another genetic screen. te Heesen and colleagues exploited the instability of this mutant at a non-permissive temperature and used a high copy number suppression approach to isolate a complex partner of Wbp1. Swp1 was therefore isolated as an allele specific suppressor of a wbp1 mutation 63. Ost2p overexpression similarly suppressed the wbp-2 phenotype 64. Since mutations of OST complex proteins can be suppressed by overexpression of other complex subunits, similar genetic approaches have been used to define all OST complex subunits. Ost5p overexpression suppresses an Ost1p mutation (Reiss et al 1997,) Ost3p and Ost4p are high copy number suppressors of stt3 mutants while Ost3p overexpression suppresses ost4p deletion phenotype 26. With the identification and cloning of all OST subunits, forward genetics strategies such as the replacement of wild type genes with altered ones, or expression of 10

22 modified genes on plasmids are continuously been used to generate OST mutants from which current knowledge is based Mass spectrometric analysis of OST Mass spectrometry is a sensitive analytical technique that allows the detection, identification and quantification of molecules in simple and complex mixtures based on their mass and charge (m/z). The sample to be measured is typically ionised and measured by an analyser. The ionised molecules are accelerated with an electric and magnetic field, separating them according to their m/z. Separated ions are detected in the detector and the results are displayed as spectra with relative abundance as a function of m/z. Identification of molecules in the sample is achieved by correlating the identified masses to known masses or by observing signature finger prints. For example, peptide-mass fingerprinting is a very simple mass spectrometric technique that can be used to identify a protein in a sample. Basically the protein is treated with proteases and masses of the peptides are measured and compared to a data base of protein masses. Every protein has a fingerprint and by matching your MS data with known peptide fingerprints one can confirm the identity of a specific peptide Shotgun proteomics for analysis of OST Shotgun proteomics is a MS technique that allows the identification of different proteins in discovery mode. A typical shotgun glycoproteomics workflow consists of glycoprotein extraction and digestion with site specific proteases such as trypsin or Lys C to generate peptides in lengths suitable for fragmentation. The peptides are separated using reverse phase liquid chromatography and ionized by electrospray ionization (ESI). Selected ions are fragmented, for example, by collision-induced dissociation (CID) tandem mass spectrometry (MS) and fragment masses are detected in a mass spectrometer producing MS/MS spectra. Comparisons of these spectra with theoretical spectra from a protein database allows the identification of proteins in biological samples 65. With respect to OST, shotgun proteomics has been used, for instance, to measure the glycoproteome of six model eukaryotic organisms and identified up to 2,254 glyco-sites in the zebrafish Danio rerio, 2,229 in Drosophila melanogaster, 2,186 in Arabidopsis 11

23 thaliana, 1,794 in Caenorhabditis elegans, 516 in Saccharomyces cerevisiae and 425 in the fission yeast Schizosaccharomyces pombe, 66. Although very powerful, this technique is limited by the speed of the instrument and sensitivity of the mass analyser. Owing to the data-dependent acquisition (DDA) nature, the stochastic selection of precursor ions for fragmentation is usually biased towards higher intensity signals. In addition, co-eluting peptides or those with similar retention times are not all fragmented due to the speed of the experiment. Together, these properties result in low or non-identification of low abundant proteins in a complex mix making it challenging to quantify a set of proteins, especially low abundant peptides reproducibly across different samples and experimental replicates 67,68. Quantitative proteomics is therefore important for precise analysis Quantitative proteomics for analysis of OST function Labelling and enrichment techniques for quantitative mass spectrometry To facilitate quantification, several labelling approaches have been developed to aid analysis including metabolic incorporation e.g. SILAC (Stable Isotope Label of Amino acids in Culture) or chemical introduction of isotopic or isobaric tags e.g. itraq (isobaric Tag for Relative Absolute Quantification) and ICAT (Isotope Coded Affinity Tag) are commonly used in quantitative MS. SILAC labelling integrates amino acids with specific heavy atoms into live cells with little or no effects on chemical properties and biology, thus, ensuring that the accuracy of quantification is not affected by lysis, fragmentation and purification when they are mixed before processing 72. It is very simple and basically involves growing different cell populations in a medium containing either light or heavy amino acids, with arginine and lysine being the most commonly used amino acids. With every cell division the amino acids are incorporated into proteins resulting in a mass shift that can be measured by MS with accuracy only affected by the signal intensity. Most researchers now couple protein-labelling strategies with glycoprotein or glycopeptides enrichment steps to reduce sample complexity and improve quantitation. Enrichment methods that target protein glycosylation primarily utilize solid-phase extraction techniques including lectin-specific capturing, hydrazide 12

24 capturing, and affinity separation approaches. The later are usually based on hydrophilic interaction chromatography (HILIC), porous graphitized carbon or immobilized boronic acid 73. Lectin enrichment assays are based on specific binding of these carbohydrate-binding proteins to unique glycan structures on glycoproteins. This property makes it possible to separate glycoforms specific to the lectin used, thereby eliminating the masking effect that other glycoforms present in the complex mix. Examples include: Concanavalin A (ConA) which binds to mannosyl and glucosyl residues of glycoproteins 74 ; wheat germ agglutinin (WGA) which binds to N-acetyl glucosamine and sialic acid 75 ; and jacalin (JAC) which is specific for galactosyl (-1, 3) N acetylgalactosamine and O-linked glycoproteins 76. However, with micro heterogeneity where glycoproteins possess different glycoforms of the same base protein, glycan structure-dependent purification may falsely isolate unrepresentative monospecies. More recently, the use of HILIC has gained popularity as a non-modifying technique for the separation and enrichment of glycopeptides in glycoproteomics. The rational is that, in a mixture of glycosylated and non-glycosylated peptides, the carbohydrate moiety renders the glycopeptides hydrophilic such that they adhere more firmly and are retained on a HILIC column separating them from the rest of the non-glycosylated peptides. The use of hydrophilic stationary phase based on function, for example, zwitterionic interaction together with relatively hydrophobic organic phases are main features of the HILIC technique 77. In 2004, Hägglund and colleagues combined the HILIC approach with enzymatic digestion to identify and unambiguously assign glycosylation sites to glycoproteins. When there is need to analyse a specific set of proteins or analytes, for example, metabolites or biomarker candidates precisely, quantitatively and reproducibly across numerous samples, targeted mass spectrometric techniques such as Selected Reaction Monitoring (SRM) and Parallel reaction monitoring (PRM) are necessary Targeted mass spectrometry: SRM SRM is a highly selective and sensitive non-scanning targeted mass spectrometric technique which allows the measurement of specific predetermined peptides or protein fragments of interest in complex biological samples. SRM is executed on a triple 13

25 quadrupole mass spectrometer where predefined ion masses of a corresponding peptide and fragment ion are selected in the first and third quadrupole while the second acts as a collision cell for fragmentation. Basically, the machine is fed with masses of several precursor/fragment ion pairs of a targeted peptide (transition list) and these masses are monitored over time generating chromatographs with signal intensities and retention times of every transition 46,78,79 (Fig. 3). The advantage of SRM over shotgun proteomics is that; the duty cycle is very high with a large dynamic range improving the identification of low abundant proteins within a complex mix that might be missed in shotgun experiments, and ensuring that multiplexed analyses are possible. In addition, the targeted nature of this method ensures high specificity and a robust measurement of a set of proteins over different conditions 67,80. However, because SRM is a targeted technique, previous knowledge of the fragmentation patterns of interested peptides is required. This means that much time needs to be invested to design an optimal assay to measure a set of peptides. In addition, the number of multiple transitions per peptide that can be measured in a single LC-MS run with high sensitivity and accuracy can be limiting in large studies 78. Furthermore, SRM does not provide sufficient sensitivity in detecting very low abundant proteins such as biomarkers or post-translational modifications in cells 81. SRM methods have improved OST function analysis and have let to the determination of OST complex assembly events Targeted mass spectrometry: PRM PRM, a data independent (DIA) technology with full scanning capabilities was designed in 2012 to circumvent the drawbacks of SRM and improve sensitivity. PRM is performed on Orbitraps or hybrid Q-Exactive spectrometers where the third quadrupole of the triple quadrupole instrument has been replaced with a high resolution, more accurate analyser that allows the parallel detection of defined ions 82 ( Fig. 3). Here, the peptide precursor ion is selected in the first quadrupole, fragmented in the second and unlike in SRM where there is a second filtration, all generated fragments are monitored in parallel with a full scan mass spectrometer. Since all generated ions are measured to confirm a peptide (instead of the maximum of 5 transitions used in SRM), PRM spectra are more specific. This also means prior knowledge of the target transitions is not required, hence, less time investment in 14

26 method development. In addition, the high resolution capacity of the instrument allows for higher sensitivity when compared to SRM, with the ability to separate coeluding background ions from those of interest 82. In this project, we use a PRM-based method to study OST activity. Figure 3: Schematic comparison of three different targeted mass spectrometric techniques. In SRM, predefined ion masses of a corresponding peptide and fragment ion are selected in the first and third quadrupole to generate chromatographs with signal intensities and retention times of every transition. In PRM, the third quadrupole of the triple quadrupole instrument used for SRM has been replaced with a high resolution, more accurate analyser that allows the parallel detection of defined ions. SWATH combines DIA with targeted data extraction on a TripleTOF mass spectrometer to increase the throughput of protein quantification Data-independent mass spectrometry: SWATH for analysis of OST SRM and PRM are still the best quantitative MS methods but are limited by the number of proteins that can be quantified in a single LC-MS run (typically ) and method development time. SWATH (Sequential Window Acquisition of all THeoretical Mass Spectra) is the most recent MS technique that combines DIA with targeted data extraction on a TripleTOF mass spectrometer to increase the throughput of protein quantification 84 (Fig. 3). During SWATH acquisition, all precursor ions within a 15

27 defined m/z- range are fragmented, and by repeatedly scanning and fragmenting through this broad defined window and retention time, full spectra of all precursors are acquired. Based on prior knowledge from proteome-specific libraries, specific peptides can then be quantified from this data by using targeted extraction methods at a level comparable to SRM, only with a higher coverage 83. Unfortunately, SWATH does not achieve the sensitivity obtained with SRM and PRM. SWATH MS approach has successfully been used to robustly measure site-specific glycosylation in human saliva with a potential clinical applications N-linked glycosylation in protein folding ER quality control and biological significance Sequential processing of N-linked glycans generates specific N-glycan structures that direct protein folding or export to degradation. N-glycans play important roles in assisting proper protein folding and increasing the stability of folded proteins. In addition, N-linked glycans in the ER function as molecular signals in the folding process and quality control 86. Newly synthesized and membrane-bound eukaryotic proteins go across the ER membrane into the lumen through the translocon complex as unfolded proteins 87. In the lumen, they fold properly, assemble into multi-subunit complexes with specific stochiometeries and are sorted and transported to their final destinations. This is possible because of the assistance of chaperons and other modifying enzymes such as those involved in N-linked glycosylation and disulphide bond formation 88,89. Even with these resources, a good amount of newly synthesized proteins fail to fold correctly and meet conformational standards. Quality control mechanisms have therefore evolved to closely monitor and eliminate misfolded proteins which might accumulate and become toxic to cells 90. Diseases caused by accumulation and aggregation of misfolded proteins such as Alzheimer s, Parkinson s and Huntington s diseases are due to failure of these quality control mechanisms emphasising their importance 91,92. 16

28 1.4.2 N-linked glycosylation: Calnexin-Calreticulin cycle Immediately after transfer of the Glc3Man9GlcNAc2 to nascent polypeptide by OST, glycan processing begins in the ER with the sequential removal of the three glycose residues by glucosidases I and II generating a Man9GlcNAc2 glycan. If the polypeptide does not properly fold, the glycan is re-glucosylated by a UDP-Glc:glycoprotein glucosyltransferase (UGT1) to re-establish the α-1, 3 glucose 93,94. The activity of this enzyme is specific for the unglucosylated glycans on unfolded but not native polypeptides making it a sensor of glycoprotein conformation. The monoglucosylated glycan is then specifically recognised and bound by the lectin-like chaperones Calnexin and calreticulin. Binding to this chaperones retains the unfolded protein in the ER and promotes its interaction with the oxidoreductase ERp57, which accelerates disulphide bond rearrangement for further folding 95. The proteins dissociate from the chaperone and pass through to the Golgi if properly folded or are immediately re-glucosylated, rebound and retained in the ER if not properly folded. Cycles of de-glucosylation and reglucosylation of the glycan by the counteracting actions of glucosidase II and UGT1 continue until proper folding of proteins is achieved. N-linked glycosylation derivatives therefore mediate glycoprotein recognition by calnexin and calreticulun which retains unfolded proteins in the ER allowing classical chaperones to enhance protein folding. Unfolded protein are targeted for degradation by the endoplasmic reticulum-associated protein degradation (ERAD) N-linked glycosylation-dependent quality control: ERAD Accumulation of misfolded proteins or components that do not incorporate into protein complexes are harmful to cells and must eventually be cleared. ERAD is a conserved ER quality control machinery comprised of multiple pathways that monitor, recognise and export misfolded proteins from the ER lumen to the cytoplasm where there are ubiquitinated and ultimately degraded by the ubiquitin-proteasome system Differentiating between folded, actively folding, and misfolded proteins is crucial for ER quality control and N-linked glycosylation plays a major role in this process. During N-linked glycosylation, OST transfers the Glc3Man9GlcNAc2 glycan to newly synthesised polypeptide. The transferred glycan is sequentially trimmed by glucosidase I and glucosidase II yielding Man9GlcNAc2. Next, a Man8GlcNAc2 glycan is generated by the action of ER mannosidase I that cleaves the middle α1, 2-linked mannose. 17

29 Several chaperons like Pdi1p and Kar2p assist the proteins to fold, most of which attain their native conformations and exit the ER 6. Irreversibly misfolded proteins sooner or later encounter Htm1p, the yeast homologue of mammalian ER degradationenhancing alpha-mannosidase-like protein 1 (EDEM1) which cleaves the terminal mannose residue from the C-branch yielding a Man7GlcNAc2. The α1, 6-mannose residue of this Man7GlcNAc2 serves as recognition signal for the lectin Yos9p 98 which together with Hrd3p and Hrd1p recognise the unfolded portions on the protein and targets it for ubiquitination 99. Misfolded proteins carrying the Man7GlcNAc2 are ubiquitinated through the sequential action of E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases. These enzymes are located in the cytoplasm, thus misfolded proteins in the lumen must gain access first to be modified. The mechanism of this event is still not fully understood. Different ERAD products are polyubiquitinated by Hrd1p or Doa10p ligases. After polyubiquitilation, N-linked glycans are removed by Png1 to generating free oligosaccharides. Finally, Rad23 binds to polyubiquinated proteins and deliver them to the proteasome for degradation Concluding remarks N-linked glycosylation is a unique protein modification pathway that regulate many aspects of protein function. This conserved modification results in the attachment of an oligosaccharide moiety onto newly synthesized polypeptide chains. Core N-linked glycosylation involves three major processes which are homologous in all domains of life albeit some variations. They include: (i) The assembly of a glycan from nucleotide activated sugars on a lipid anchor by the sequential action of several glycosyltransferases. Synthesis begins in the cytoplasm and the LLO is flipped to the lumen of eukaryotes or periplasm of prokaryotes where it can be further extended and transferred to a newly synthesize polypeptide. (ii) Peptides with N-X-S/T sequons where X can be any residue except proline are acceptor substrates of the OST (iii) The fully assembled glycan is transferred en bloc to asparagines of the glycosylation sequon by an oligosaccharyltransferase. In the ER, glycans play a fundamental role in protein folding and quality control. By serving as universal tags, glycans allow specific interactions with lectins and modifying enzyme that promote folding or target misfolded proteins for degradation. The biosynthesis of the glycan is therefore tightly regulated to ensure that only completely assembled glycans are primarily transferred 18

30 to proteins 23. Glycan processing in the Golgi generate a diversity of structures that are displayed on cell surfaces with new sets of functions. This separation of synthesis and processing in different cellular compartments is an adaptation that favours efficient exploitation of glycan function. In bacteria, the N-X-T/S sequon is extended to contain an aspartic or glutamic acid in the -2 position relative to the glycosylated asparagine thereby restricting glycosylation to a limited set of peptides when compared to its eukaryotic counterpart. Eukaryotes on the other hand have a wide range of peptide substrate and have evolved strategies that allow efficient and maximum glycosylation. Higher eukaryotes have added other subunits to the catalytic Stt3 protein forming complexes of up to eight subunits while lower eukaryotes have duplicated the Stt3 protein. Ultimately, the proteins glycosylated by prokaryotes and eukaryotes are vastly different despite the high similarity between the N-linked glycosylation pathway in both domains of life. 19

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42 Chapter 2: Insights into the function(s) of Saccharomyces cerevisiae Stt3 protein: A structure-function characterization. Elsy Ngwa, Kristina Poljak, Chia-Wei Lin, Susanne Mueller and Markus Aebi Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology, ETH Zurich, CH-8093 Zurich, Switzerland Contributions Construction of strains Preparation of samples for mass spectrometry Planning and execution of experiments PRM Method development SRM, PRM and shotgun Mass spectrometric measurements Data analysis and writing of manuscript 31

43 Summary The yeast Stt3 protein is the catalytic subunit of the eight-subunit protein complex of the oligosaccharyltransferase (OST). This enzyme is responsible for N-linked glycosylation, a highly conserved and essential posttranslational protein modification in eukaryotes that generates a huge diversity of glycoproteins with diverse cellular functions. The function of this essential Stt3 protein is still not fully understood. We used mutagenesis to elucidate its function based on structure by looking at the effects of mutations on OST complex formation and N-glycosylation of proteins in vivo. Point mutations were generated at the chromosomal level in yeast cells harbouring the complementing Leishmania major STT3 (LmSTT3D) protein using site-directed mutagenesis. The effect of the mutations on enzyme activity was evaluated in strains without the complementing LmSTT3D protein. Phenotypic analysis of the mutant strains was performed with respect to formation of the octameric complex and catalytic activity. The role of stt3p mutants in complex formation were investigated by monitoring OST subunit turnover rates and steady state levels of subunit proteins. Glycosylation efficiency of mutants was determined by analysing site-specific occupancy for different substrate proteins by PRM mass spectrometry. We defined residues that are essential for catalysis and are proposed to form the catalytic centre of OST. Mutations of these residues altered N-glycosylation but not complex formation. In contrast, other mutations induced rapid degradation of some OST subunits, indicating complex destabilization. This led to reduced enzyme activity and hypoglycosylation of proteins. More interestingly, some mutations outside the putative catalytic centre do not destabilize the complex, but greatly reduced enzyme activity. 32

44 Introduction N-linked glycosylation is the most ubiquitous and complex post-translational modification of a majority of eukaryotic secretory and membrane-bound proteins. This ER localized pathway, which acts as an essential determinant for molecular recognition specificity as well as protein folding and stability, is essential for cell viability and is evolutionarily highly conserved 1 5. The central enzyme in this pathway, the oligosaccharyltransferase (OST), catalyses the transfer of a dolicolphosphate-linked oligosaccharide (LLO), Glc3Man9GlcNAc2, onto selected asparagine residues in the sequon Asn-X-Ser/Thr of newly synthesised polypeptide chains translocating into the ER. In the yeast Saccharomyces cerevisiae, the OST is a multi-subunit membrane-bound complex composed of nine proteins; OST3, OST4, OST5, OST6 OST1, OST2, WBP1, SWP1 and STT Of these, OST1, OST2, WBP1, SWP1 and STT3 are essential for cell viability, while the others, though not essential are needed for maximum enzyme activity. The roles of the OST subunits and how they interact with each other are still not fully understood, but there is reliable data showing that Ost3 and OST6 which incorporate into different OST complexes have oxidoreductase activity. They help with efficient glycosylation of translocated polypeptides by directly affecting their oxidative folding A combination of photolabeling and mutagenesis studies in yeast and mammalian OSTs suggested that STT3 is the catalytic subunit harbouring the peptide recognition site and responsible for the transfer of the glycan 7,8,12,13. More convincing evidence of its catalytic role came from the discovery of bacterial and archaeal homologues PglB and AglB respectively, which catalyse N-linked glycosylation as single proteins in the absence of other subunits 14,15. In mammalian cells, two homologues of STT3 (STT3A and STT3B) are expressed and incorporated into distinct complexes with the other subunits N33/Tusc3 and IAP3, ribophorin I, DAD1, OST48, OST4 and ribophorin II. These distinct isoforms show different activity profiles, and have the ability to discriminate between donor substrates with different oligosaccharide structures 16. KCP2 and DC2 have been identified as potential new subunits of mammalian OST 17. Stt3p is the most conserved of the OST subunits, and that of S. cerevisiae shares a 60% sequence identity and 80% similarity to the human homologue 12. Despite the essential role that this subunit plays in N-linked glycosylation, its mechanism of action in eukaryotes and the manner of interaction 33

45 with other subunits remain obscure. To date, no high resolution structure exists for the eukaryotic Stt3 proteins. High resolution structures, however, exist for the bacterial and archaeal homologues, and have led to a proposed mechanism for the glycosylation reaction in prokaryotes 18,1920. Nonetheless, such a mechanism cannot be generalized and accepted as universal in eukaryotes without validation. In this study, we took advantage of this knowledge and used mutagenesis studies to further elucidate the function of S. cerevisiae Stt3p. We report the identification and characterization of stt3p point-mutations and their effects on N-linked glycosylation of yeast proteins and the stability of the OST complex. We show specific residues that are involved in direct catalysis either by coordinating the metal ion in the catalytic centre of Stt3p or interacting with the pyrophosphate of the lipid-linked carbohydrate substrate. In contrast to these residues that abolish N-linked glycosylation but do not affect complex stability, we also report other novel mutants that destabilize the OST complex and alter glycosylation by inducing rapid degradation of some OST complex proteins. 34

46 Experimental strategy and procedures Media and growth of strains Yeast strains used in this study are described below and listed in table S1. Standard genetic methods and yeast media 21 were used to cultivate yeast cells. To maintain plasmids within cells, the cells were grown on synthetic media lacking the appropriate amino acid needed for selection. The lithium acetate method was used for transformation of yeast cells 22. Construction of strains and genetic techniques arg4 deletion We started with the Euroscarf BY4742 strain with genotype Matα, his3δ1; leu2 Δ0; lys2 Δ0; ura3 Δ0. To allow Stable Isotope labelling of Amino Acids in Cell culture (SILAC) followed by MS analysis, the ARG4 gene was completely deleted using the loxp/cre system 23,24. This system combines the advantage of using a gene disruption cassette, in this case KanMX, with those of Cre/loxP recombination. KanMX flanked with two direct repeats of loxp sites and homologous DNA sequences from ARG4 was transformed into our yeast cells (Fig.1A). Transformants with correct integration of cassette and concomitant deletion of Arg4 were confirmed by PCR. The resulting yeast strain was transformed with psh47, a URA3 plasmid that carries Cre recombinase under the control of GAL1 promoter 23. Expression of Cre recombinase by shifting cells from a glucose to galactose medium resulted in the removal of the KanMX cassette leaving behind only one loxp site at the original chromosomal locus. Cell growth on medium containing 5-Fluoroorotic acid (5-FOA) allowed for selection of cells that had lost the URA3 plasmid, thereby getting rid of the psh47 plasmid. The final strain generated from this deletion; KP4(Matα arg4 0; his3 ; lys2 0; ura3 0) is henceforth referred to as wild type in this study and used for further analysis. 35

47 Figure 1: Strategy for construction of stt3p mutants. (A) ARG4 knockout with loxp-kanmx-loxp cassette. Cre recombinase allows for recombination between the two-loxp sites resulting in a pop out of the kanmx cassete leaving behind one loxp site. (B-C) lethal deletion of yeast STT3 complemented with the Leishmania major paralogue LmSTT3D after which the endogenous copy was deleted. (D) STT3 was amplified from genomic DNA bearing a homologous recombination sequence 200bp downstream of the STT3 gene so as not to affect its terminator while KanMX was amplified from pfa6-kanmx plasmid. Linearized prs313 and the separate PCR products homomogously recombined to generate STT3-KanMX plasmid. (E-F) Point mutants were generated by site-directed mutangenesis and reintegrated into original locus. G) General genotype of all stt3 mutants analysed. 36

48 Stt3p deletion As highlighted in the strategy above (Fig.1C), yeast STT3 gene was knocked out using the LEU2 gene from Kluyveromyces lactis 25. The PCR product generated contained the LEU2 gene flanked by base pairs of DNA homologous to the yeast STT3 locus. Due to the high efficiency of recombination in yeast, transformation of yeast cells with this PCR product resulted in the replacement of STT3 with the LEU2 cassette. Cells bearing correct replacement grew on a selective medium lacking Leucine and were further confirmed with PCR and DNA sequencing. This deleterious loss of functional STT3 was complemented by expressing Leishmania major STT3D protein, which has previously been shown to complement the deletion of the endogenous copy 26 (Fig.1B). The strain generated after this deletion is called EN0 with genotype Matα stt3::leu2 arg4 0; his3 ; lys2 0; ura3 0. Construction of Stt3p plasmid The main purpose of our study was to elucidate the function of yeast STT3 using chromosomally generated mutants. Site-directed mutagenesis on plasmid was the easiest way to generate such mutants, so we cloned a plasmid carrying yeast STT3 in frame with a KanMX marker gene for selection purposes, using recombinationassisted PCR targeting technique. This was possible owing to the observation that linearized DNA fragments readily and efficiently trigger recombination and in vivo ligation in S. cerevisiae 27. We generated two separate PCR products having the yeast STT3 (amplified from yeast chromosomal DNA) or KanMX cassette (amplified from the plasmid pfa6-kanmx4) with colour coded primers as shown in Fig. 1 D. A double digest of the yeast plasmid prs313 28,29 with restriction enzymes XbaI and XhoI generated a linear plasmid DNA with ends homologous to regions of the STT3 and KanMX PCR fragments. Co-transformation of yeast cells with the PCR fragments and linearized plasmid DNA resulted in recombination, generating the STT3-KanMX plasmid (Fig. 5D). Transformants were grown on full medium overnight at 30 C and replica plated on medium containing geneticin (G418) the following day. Because KanMX confers resistance to the drug G418, only cells that had correctly assembled the plasmid could grow. The plasmids were recovered from yeast as described 30 and 37

49 amplified in E. coli. Re-isolated plasmids from the E. coli were doubly digested with restriction enzymes and sequenced to confirm generation of the correct plasmid, which served as a platform for site directed mutagenesis. Generation of stt3 mutants Using a Phyre2-based structural alignment, we selected amino acid residues that were speculated to form the putative catalytic site of Stt3p or interact with the LLO donor substrate and mutated them on the STT3-KanMX plasmid. Additional amino acid residues were also selected based on their conservation amongst OSTs from several species. Stt3 mutants were generated using the Phusion site-directed mutagenesis protocol from Thermo Scientific. All primers used for mutagenesis and strain construction are listed in appendix 1. Mutagenesis primers where designed such that they annealed back-to-back to the plasmid, one having the desired mutation and the second not. These primers where phosphorylated and used to PCR- amplify the STT3- KanMX plasmid. The mutated PCR product was circularized by ligation with T4 ligase and the product was transformed into Chemo-competent E. coli DH5α using standard protocol 31. Cells were selected on Luria broth (LB) medium 32, supplemented with 100μg/ml ampicillin, and mutations were confirmed by sequencing. Mutant stt3 proteins were amplified and reintegrated into the original chromosomal locus through homologous recombination (Fig.1F and G). In the presence of the complementing LmSTT3D protein, we analysed the effect of a mutation on assembly of the OST complex and in its absence, we assayed for growth and glycosylation efficiency of mutant compared to wild type yeast. All strains and plasmids used and generated in this study are listed on table S1 and plasmids on table S2. Yeast transformation We used the high-efficiency lithium acetate method 22 to transform yeast cells with either 5µg of linear PCR product or 50ng of plasmid DNA. For auxotrophic mutants resulting from a plasmid transformation, transformants were selected directly on dropout medium. However, for reintegration of the G418-resistant Stt3-KanMX construct, the cells were allowed to recover overnight on a non-selective medium (YPD) and replica plated on G418-containing medium the following day. Single colonies from 38

50 transformation plates were re-streaked and grown twice to select for pure isolates. The pure isolates where then grown on 5FOA for several days after which they were tested for their ability to grow on medium lacking uracil. Phyre2-based structural prediction Protein structure is more conserved than protein sequence and only a limited number of unique protein folds exist in nature 33. Based on this dogma, the structure of a known protein sequence can be predicted by comparing it to a database of known structure with fair confidence. We used the free online Phyre2 portal, which is amongst the most used structure prediction tools, to predict the structure of yeast Stt3p based on its primary amino acid sequence 34. Phyre2 compares a selected sequence to a large library of sequences, to generate an evolutionary and statistically significant profile, which is subsequently scanned against a library of profiles with known structures. The resulting sequence alignment between the unknown and known structure is used to build a model of one sequence or structure based on the known structure (Fig.2). One of the top-scoring model of our Phyre search, with yeast STT3 as query sequence was based on the structure of its bacterial homologue, the PglB. In this model, 97% of the amino acid residues were modelled at >90% confidence. Unfortunately, the drawback we encountered when using Phyre2 (a property shared by all other online prediction tools), is that regions with no homology between the yeast STT3 sequence and that of the PglB structure could not be modelled. For example, 26 residues are inserted in the N-terminal region of the EL5 of yeast Stt3p between residues 309 and 335 making it longer than that of the PglB and were not modelled in the Stt3p structure. 39

51 Figure 2: Schematic representation of Phyre2 work flow showing the different algorithms. (1) shows generation of homologous sequences from different databases to predict secondary structure of query sequence. (2) models from 1 are scanned against a database of known structures. (3-4) the best scoring alignment is used to generate the final Phyre2 model (Kelley LA et al.2015). Analysis of Stt3p mutants OST complex stability and turnover by SILAC-SRM mass spectrometry The subunits of the yeast OST complex encoded by OST3, OST4, OST5, OST1, OST2, WBP1, SWP1 and STT3 are located on three putative sub-complexes. In the past, Stt3p mutants with severe phenotypes were usually a result of complex destabilization. We analysed the stability of the OST complex after introducing single amino acid changes in Stt3p and complete and partial deletions of the luminal domain. The questions we addressed where; (1) is a reduction in OST activity always as a result of complex destabilization? and, (2) are there residues that affect enzyme activity but not complex stability? If yes, what do they do? We assumed the OST complex is stable in wild type 40

52 yeast with all subunit proteins present in similar amounts at a stoichiometric level 35, ensuring a hundred percent glycosylation of proteins. However, a perturbation could lead to destabilization of some or all subunit proteins resulting in a faster degradation, with less amounts of such proteins within the complex. This would result in destabilization of the whole complex with complete loss of glycosylation or just hypoglycosylation of some proteins (Fig. 3). As such, we could analyse for complex stability in two ways: either looking at the amounts of OST proteins within the complex at steady state, or analysing the degradation rate of OST proteins during exponential growth. Figure 3: Schematic representation of the effect of a point mutation on OST complex stability. A. Stable active OST in wild type with normal N-linked protein glycosylation. B. A mutation of stt3p causes faster degradation of Ost3/6 resulting in destabilisation of complex, severe hypoglycosylation or lethality. Analysis of relative amounts of OST subunits in steady state We used the SILAC pooling protocol previously described to analyse relative amounts of OST proteins in steady state 35. Wild type yeast was grown in medium containing heavy arginine and lysine, while the OST point mutant to be analysed was grown in medium containing light amino acids. Heavy and light-labelled cells were pooled together in a one-to-one ratio of their weights and MS samples were prepared from the pool as described below (Fig. 4). The abundance of subunit protein in mutant stt3p relative to the wild type was calculated from the light-to-heavy ratios of OST peptides normalized to the average L/H value of the ribosomal protein Rpslap. Precisely, we log2 transformed L/H ratios for all peptides and averaged the log values over all peptides of a protein. Log-transformation ensured that the 41

53 variation in the data was similar across orders of magnitude bringing data closer to normal distribution. We then normalized these values by dividing by the log2 values of the ribosomal control protein Rps1ap of the same replicate. Results for three biological replicates were averaged and back-transformed to linear space. Figure 4: Schematic representation of steady state SILAC analysis of OST complex stability. Yeast stt3 point mutant was grown in light amino acid-containing medium and the wild type in heavy amino acid containing medium. MS samples were prepared for equal weight pooled samples and measured through SRM MS. Relative protein amounts were calculated from L/H ratios of each peptide. Reduction in subunit protein in mutant relative to wild type implied complex destabilisation. Adapted from Mueller et al., Analysis of OST complex-protein turnover We analysed OST-subunit protein degradation rates by using SILAC pulse-chase experiments 35. Briefly, arginine and lysine auxotrophic yeast mutants were grown in dropout medium (SD-arg-lys) supplemented with 20mg/l of heavy 13 C6 arginine (R6, Cambridge Isotope Lab, Andover, USA) and 15 N2 lysine (K8, Sigma Aldrich, Buchs, Switzerland) isotopes. At OD600 1, the cells were collected on a 0.22µ filter and washed with 400ml of SD-arg-lys medium. The cells where then resuspended in 200ml of medium containing same concentration of light 12 C6 (Ro) arginine and 14 N2 (K0) isotopes, and chased for two hours until all newly synthesised proteins incorporated the light amino acids. At three time points (0, 1hr, 2hrs), 50 ODs of cells where collected, from which proteins where isolated and analysed by SRM (Fig. 5). Calculation of degradation rate was done by normalising the rate of loss of old proteins from the cell to the dilution of protein content by cell division using the protein Rpslap as a stable reference 35,36. Ln (2) of the degradation value provided the half-lives of the 42

54 OST proteins monitored. Proteins with half-lives greater than 12 hours (8 cell divisions) were considered stable, otherwise they were considered instable. Figure 5: Schematic representation of protein-turnover rate experimental set-up. Heavy arginine and lysine-labelled yeast mutants were moved to medium with light amino acids. At different times points, cells were sample for MS sample preparation. The changing heavy -to -light ratios of OST peptides were monitored by SRM on the ABSIEX QTRAP5500 mass spectrometer. Degradation rate was calculated from the heavy- to -light ratios of related peptides. Isolation of microsomal proteins for SRM analysis. Microsomal proteins were isolated from membrane-enriched samples as described previously 35. 5oODs of cells were lysed in 2M NaCl, 10mM 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES)/NaOH, ph 7.4, 1mM EDTA, and protease inhibitors (Complete; Roche Diagnostics International AG, Rotkreuz, Switzerland) with glass beads at 4 C for 15 mins. After centrifugation at 1000 g for 5mins, the cleared lysate was collected and subjected to further centrifugation at 16,000 g at 4 C for 20 mins to collect the microsomal fraction. Membranes from vesicles were linearized by resuspending and incubating the microsomes in 1ml of 0.1 M Na2CO3 and 1mM EDTA, ph 11.3, at 4 C for 30 mins. The suspension was centrifuged and the microsomes in the pellet fraction were washed three times, first with 1 ml 5M urea, 100mM NaCl, 10mM HEPES, ph 7.4, and 1mM EDTA, and then two times with 1ml 0.1M Tris/HCl, ph 7.6. Microsomes where then solubilized in 100µl of detergent containing 4% SDS, 50mM dithiothreitol (DTT), and 0.1M Tris/HCl, ph 7.6. Following centrifugation, proteins collected in the supernatant were further processed following the filter-assisted sample preparation protocol 37. The solubilized samples were 20- times diluted with UA buffer (8 M urea, 0.1 M Tris/HCl, ph 8.5) and applied to a 30- kda cut-off filter device (Amicon; Millipore, Billerica, MA). The columns were washed 43

55 with 0.4ml UA and samples were alkylated by incubating with 0.2ml of 50 mm iodoacetamide for 30 mins in the dark. After alkylation, the proteins were then washed with 0.4ml of buffer UB (8 M urea, 0.1 M Tris/HCl, ph 8.0. Following three washes, the proteins were resuspended in 60μl UB and digested for 16hrs with 2μg of endoproteinase Lys-C (Wako Pure Chemical, Richmond, VA) diluted with 400μl of 40mM NaHCO3. A second digestion was done by incubating the proteins with 2μg of trypsin for 4hrs at 37 C. Peptides were eluted by centrifugation and 5μg of the eluded peptides were diluted to a final concentration of 3% acetonitrile and ph 2 3 with trifluoroacetic acid (TFA). The samples were desalted over C18 ZipTips (Millipore, Zug, Switzerland) and dried in the speedvac. Samples were dissolved in 20μl of 3%ACN and 1% FA and Retention time irt peptides (Biognosys, Schlieren, Switzerland) were added in a 1:40 ratio. 2 8 μl of final sample was analysed by mass spectrometry. Mass spectrometry analysis SRM analysis was performed on a QTRAP 5500 (AB Sciex) supplied with a nanospray ion source. The Interface temperature was set at 170 C, ion spray voltage V, ion source gas pressure, 6 10 psi, curtain gas pressure 25 psi, and collision gas set to high. The declustering and collision cell exit and entrance potential were set, at 100, 13 and 10 respectively. The retention time windows for the transitions, and a target scan time were set for 5 mins and 3s respectively. Scheduled SRM results were exported to skyline daily, an open source software for quantitative proteomics analysis 38. Every individual peptide peak was manually checked for correct assignment and accurate integration. 44

56 Glycosylation site occupancy Analysis of N-link glycosylation efficiency of yeast proteins by Immunoblotting The glycosylation efficiency of OST was analysed by evaluating the glycosylation pattern of two OST proteins: Ost1p and Wbp1p (Fig. 6A) and the vacuolar glycoprotein CPY (Fig. 6B) using a previously described protocol 35. Briefly, we lysed exponentially grown cells (OD ) with glass beads in sample buffer by vortexing for 15 mins at 4 C. The sample buffer contained 2% SDS, 62.5 mm Tris/HCl, ph 6.8, 10% glycerol, 6 M urea, 5% β-mercaptoethanol, 0.02% bromophenol blue, protease inhibitor (Complete; Roche), 5 mm phenylmethylsulfonyl fluoride, 25 mm EDTA. Protein concentrations were measured with the Pierce BCA assay (Thermo Scientific, Rockland, USA) before adding bromophenol blue. Upon addition, samples where incubated for 20 mins at 37 C. Same protein amounts were separated on PAGE gels and transferred onto nitrocellulose membranes. Membranes were blocked with 5% milk in PBS-tween, hybridized with primary antibodies against target proteins, and finally secondary antibodies. Membranes were developed for visualization with ECL solution (GE Healthcare, Amersham) and light-sensitive films (Super RX, Fuji Medical X-Ray Film; Fuji, Tokyo, Japan). Ost1p, Wbp1p and CPY are glycoproteins with four, two and four glycans respectively. In wild type yeasts where OST is fully functional, all sites are glycosylated and are visualised as a single band on immunoblots. However, reduced enzyme activity caused by a mutation results in hypoglycosylation generating different hypoglycosylated forms that depend on the severity of the perturbation (Fig. 6). 45

57 Figure 6: Example of immunoblot analysis of glycosylation efficiency. In wild type yeast, OST activity is 100% and all proteins are glycosylated and visualized as a single band on immunoblots. With a reduced OST activity; some sites are not glycosylated for example in (A), one of the two sites of Wbp1p was not glycosylated, while in (B), none of the four sites of CPY was occupied. Analysis of systemic N-linked glycosylation efficiency by SILAC- PRM mass spectrometry In a SILAC set-up, wild type cells were grown in heavy medium and stt3p mutants were grown in light medium. From 100 OD of yeast cells, ER proteins were isolated from membrane-enriched samples as described above for the SILAC pooling experiment. After digestion of proteins with Lys-C and trypsin, the eluded peptides were dried and resuspended in 500µL of 50mM NaOAc, ph µ of Endoglycosidase H (Endo H) was added and incubated overnight at 37 C. Another microliter of Endo H was added to the sample and incubated for four more hours to allow for efficient digestion. The sample was desalted over C18 Sep-Pak cartridge (Waters Corporation Milford, Massachusetts USA). To do so, 50mg Sep-Pak C18 cartridge was conditioned with 1mL of CAN, then equilibrated with 1 ml of 60% ACN and 0.1% TFA. After washing the equilibrated column with 2ml of 0.1% TFA, the Endo H- digested sample was loaded onto it. The column was washed with 6 ml of 0.1 % TFA and bound peptides were eluted with 1 ml of 60% ACN and 0.1% TFA and dried. The sample was resuspended in 100µL of 3%ACN and 1% FA and irt peptides were added 0.25µg peptides and analysed by mass spectrometry. Results were displayed as glycosylation site occupancy for every individual site relative to the wild type control (Fig. 7). 46

58 Analysis was done on the Q Exactive HF mass spectrometer equipped with an ultrahigh-field Orbitrap analyser which doubles its speed and resolution while maintaining high spectral quality for sensitive detection and confident quantitation of peptides. A samples was injected into the analytical C18 column of the Q Exactive that has an integrated HPLC coupled with a nanoelectrospray ion source (Thermo Fisher Scientific) and operated at KV. For some peptides, a full mass spectrum at 30,000 resolution relative to m/z 400 at a maximum fill time of 45ms was set (appendix 2) while for others from very low abundant proteins, resolution was set at relative to M/Z 400, maximum fill time 110 msec (appendix 3). Fragmentation was done at a normalized collision energy of 28 with target ion values of 1e 6. The Q Exactive was operated in the targeted MS/MS mode, with Xcalibur software, a scheduled inclusion list with a retention time window of 10 mins generated using skyline. The inclusion list consisted of m/z of precursor peptides of interest and corresponding retention times with at least two peptides representing a protein. Using linear regression curves, instrument spectra mass accuracy was calibrated before measurements, constantly checked and recalibrated (approximately after 2 days) during long run periods. All integrated peaks were manually curated and product ions that showed interfering signals or did not match the retention time of the other monitored co-eluting ions were excluded from quantification. Figure 7: Schematic representation of MS-based method for global analysis of glycosylation site occupancy of yeast proteins. Wild type yeasts grown in heavy medium is mixed in a one -to -one ratio with stt3p mutant grown in light medium. From the pooled samples, proteins were digested and the glycans cleaved leaving behind a peptide decorated with a single GlcNAc residue which could be identified in a targeted MS measurement. Relative quantification of every glyco-site provided occupancy values for the mutant relative to the wild type. 47

59 Calculation of glyco efficiencies of stt3p mutants The glycosylation site occupancy of stt3p mutants relative to wild type was calculated from the light-to-heavy ratios of measured peptides. Basically, we log2 transformed L/H ratios for all peptides of proteins analysed. The log2 values of all control proteins (RpI5p and Rps1ap) and non-glycosylatable peptides were averaged over all peptides of a protein. All peptides were normalized to the average of the control proteins by dividing the log2 values to those of the average of the control proteins. All glycopeptides were similarly normalised to their corresponding non-glycosylatable peptides. Results for three biological replicates were averaged and back-transformed by deloging to linear space and expressed as percentages. Sample preparation with glycopeptide enrichment for PRM method development From 100 OD of wild type yeast cells, ER proteins were isolated from membraneenriched samples as described above for the SILAC pooling experiment. After digestion of proteins with Lys-C and trypsin, the samples were enriched for glycopeptides. This was done to reduce sample complexity and to improve the identification of as many glycopeptides as possible, used for the development of the PRM inclusion list. Enrichment of glycopeptides from wild type yeasts was done using the ZIC-HILIC protocol previously described 39. Basically, the ZIC-HILIC micro column (SPEcartridge: SeQuant ZIC-HILIC 1mg, Sequant (Umeå, Sweden) was washed with 3mL of 99.5% H2O, 0.5%FA and equilibrated with 6mL of 80% ACN, 0.5% FA. SpeedVacdried trypsin-digested sample was dissolved in 500uL 80% ACN, 0.5% FA and loaded onto the column. The column was washed three times with 5mL 80% ACN, 0.5% FA. Bound peptides were eluted with 5mL of 99.5% H2O, 0.5% FA and both the flowthrough and elution fractions were collected and dried in the Speedvac. Dried samples where resuspended in 200ul 50mM sodium acetate (NaOAc), ph µ of Endo H was added and incubated overnight at 37 C. Another microliter of Endo H was added to the sample and incubated for four more hours to allow for efficient digestion. Sample was desalted over C18 Sep-Pak cartridge (Waters Corporation Milford, Massachusetts USA) as described above. The sample was dried, resuspended in 100µL of 3%ACN and 1% FA and irt peptides were added before mass spectrometric analysis. 48

60 Shotgun proteomics on Q Exactive HF followed by a Mascot (Matrix Sciences Ltd., Cheshire, UK) search let to the identification and selection of target glycopeptides with good MS properties. The following search parameters were used; oxidation of methionine residues, carbamidomethylation of all cysteine residues, 1 missed tryptic cleavage site, a 0.6 Da error tolerance in both MS and MS/MS, a 5ppm peptide tolerance, and a HexNAc (CID/HCD) modification of an Asparagine residue. All modified asparagine residues were manually validated on the produced spectra to ensure that it was found within the consensus sequon NXS/T. On skyline, optimal transitions from the Mascot file suitable for PRM assay development were extracted and used to generate a peptide library. PRM method development 60 glycosites within 40 glycoproteins were selected from the search above and validated for the PRM assay on the Q Exactive HF (The Thermo Scientific, Bremen, Germany) mass spectrometer. For every glycoprotein included in the PRM assay, 2-3 proteotypic non-glycosylatable peptides were also selected together with peptides of the ribosomal proteins RpI5 and Rps1a for protein expression normalization purposes. For some peptides, transitions were selected from the shotgun run on the Q Exactive HF described above while for others, the transitions were extracted from Picotti's yeast spectra library _QQQ_lib.tgz 40. The retention times of peptides were extracted from the unscheduled shotgun run. All transitions/precursors were analysed for proteins, and spectra peaks were manually curated to ensure correct assignment and accurate integration. Correct precursor peaks were those in which transitions coeluted together at the same retention time, showed relative intensities for light and heavy precursors in SIILAC state, and had a dot product > when compared to the ion library spectra. The 5 most intense transitions with precursors having the least background or interference were selected for the PRM method. Validation was done by determining the presence and amounts of these sites across different sample preparations and labelling states in SILAC mixing experiments with no glycopeptide enrichment. The method was finally used to analyse the glycosylation site occupancy of stt3p mutants as described above. 49

61 Analysis of growth Yeast cells carrying the reintegrated mutation and LmSTT3D-URA3 plasmid were grown on medium containing 5FOA. URA3 gene encodes an enzyme that decarboxylates 5FOA to a very toxic metabolite, 5-fluorouracil 41. Therefore, the presence of 5-FOA allowed the selection of cells that have lost the LmSTT3D-URA3 plasmid. The phenotype of the resulting URA minus cells depended on the function of the mutant stt3 protein (Fig.8). All viable cells were shifted from the 5FOA-containing medium to full medium (YPD), and pure isolates from these plates were tested for growth on YP glycerol to eliminate mutants that were petite (frequently arising due to repeated selection procedures). The non-peptite isolates were grown on YPD at temperatures between 23 C to 37 C, to test for temperature sensitive growth. Figure 8: Principle of 5FOA growth assays. Stt3p mutants complemented with URA3-plasmid bearing LmSTT3D protein were grown on medium containing 5FOA. A mutation could have no relevance on enzyme activity with normal cell growth (8.1), or reduce enzyme activity thereby reducing growth (8.2) or completely inactivate the enzyme resulting in lethality (8.3). Some viable mutants can however, have reduced fitness, and become temperature sensitive at 37 C (8.4). 50

62 Results Identification of S. cerevisiae amino acid residues for mutagenesis The aim of this project was to generate point mutations on yeast STT3 chromosomal DNA and to study their effects on N-linked glycosylation of proteins, and on OST complex formation in vivo. The high resolution full-length structures of the STT3 homologues from bacteria (Campylobacter lari) and archaea (Archaeoglobus fulgidus) have shown that the enzymes are very similar structurally despite sharing a low sequence identity 18,19. We hypothesized that the eukaryotic Stt3p also shares a similar architectural conservation and mechanism of action. As such, we used a model of yeast Stt3p based on the structure of the PglB (Fig. 9A) to identify Stt3p residues for targeted mutagenesis. The PglB structure was more suitable because its in-depth characterization suggested the mechanism for the glycosylation reaction. The positions of the amino acid residues of stt3p mutated are shown on the modelled structure (Fig. 9B). Figure 9: Structure of Oligosaccharyltransferase. (A) Crystal structure of PglB from Campylobacter lari showing bound acceptor peptide in yellow as a stick representation. The transmembrane domain is shown in blue and periplasmic domain in green Lizak et al (B) structural model of yeast Stt3p showing acceptor peptide in yellow and the peptide binding WWDY motif in blue. Residues mutated and described in this study are shown in red. 51

63 Mutations of catalytic residues inactivate OST but do not affect complex stability The structure of the PglB solved in the presence of an acceptor peptide highlighted some important observations on the catalytic mechanism of OST. In view of a common overall structural similarity and catalytic site architecture between the archaeal and bacterial OSTs, we mutated stt3p residues corresponding to the catalytic amino acid residues of PglB. We analysed the effect of these mutations on growth as a readout for enzyme activity in vivo. To do so, we introduced point mutations into the STT3 locus in the presence of the complementing Leishmania major STT3D paralogue. We subjected these cells to growth on medium containing 5FOA where survival depended on the ability of the enzyme to retain all or some enzyme activity. Mutations of residues D166, E168 and E350 to any other amino acid abolished the enzyme activity as shown by the inability of the strains to grow on 5FOA medium (Fig. 10A). On the contrary, mutation of the yeast residue N61 which should correspond to the D56 catalytic residue had no effect on enzyme activity and growth. These results indicated that N61, the only of the four residues that is not conserved amongst eukaryotic OSTs, was not involved in the coordination of the divalent metal ion and is not part of the catalytic centre. To determine the 4 th residue involved in metal ion coordination, we structurally aligned Stt3p to PglB as opposed to the amino acid sequence alignment previously used for the identification of N61. This alignment let to the identification of residue D47 as the corresponding PglB D56 residue. Mutation of D47 to an alanine did not support growth on 5FOA medium indicating an essential function of this residue in OST activity. The results suggested that residues D47, D166, E168 and E350 form the catalytic site of yeast Stt3p and coordinate the metal ion. Arginine 404 was identified as a potential residue involved in the binding of the phosphate group of the dolichol phosphate sugar forming the putative carbohydrate-binding site. The location of this positively charged residue at the interface of the transmembrane and luminal domains in close proximity to the catalytic centre supported our theory of it being a potential phosphate-binding residue. We mutated this residue and analysed its effect on enzyme activity and growth. Mutation of R404 to an alanine or to a negatively charge glutamate completely abolished enzyme activity resulting in lethality. In contrast, R404K retained some enzyme activity and supported growth on 5FOA-containing medium. 52

64 Figure 10: Mutation of residues directly involved in catalysis inactivate OST but do not destabilize complex. Yeast strains containing a single point mutation on the stt3 protein were complemented by a URA3 plasmid encoding the Leishmania major STT3D protein. Serial dilutions of mutants were spotted on medium containing 5FOA and incubated at 23 C for several days to select for ura - cells. Mutations that inactivate the enzyme did not support growth of 5FOA (A). Serial dilutions of the point mutants that grew on 5FOA were spotted on YPD medium to check for temperature sensitivity (B). Yeast stt3 point mutants were grown in light amino acid-containing medium and wild-type in heavy amino acidcontaining medium. MS samples were prepared from equal-weight-pooled cells and measured by SRM. OST subunit protein abundance relative to wild type are shown for mutants (C). Asterisk indicate OST subunits that are degraded upon deletion of stt3p. Data shown represent 3 biological replicates. 53

65 Growth on YPD medium showed that R404K functioned at a much reduced rate and that this activity was completely lost at an elevated temperature of 37 C, resulting in a complete temperature sensitive phenotype (Fig.10B). R404K severely hypoglycosylated yeast proteins at 30 C as shown in Fig. S1 A and B confirming the importance of this residue for maximum glycosylation efficiency. We next examined the effect of these point-mutations on OST complex stability by monitoring steady state levels of OST proteins. Since these residues were essential for cell viability, we took advantage of the plasmid borne LmSTT3D protein. This single subunit OST has been shown to complement the loss of endogenous activity by glycosylating most yeast proteins without integrating into the complex, even upon deletion of one or more OST subunits 26,35. Therefore, in the presence of this plasmid, stt3p point-mutants were viable and mutable for complex stability analysis. We performed SILAC experiments, where wild-type cells grown in medium supplemented with heavy isotopes of arginine and lysine were pooled in a one-to-one ratio with stt3p point-mutants grown in medium with light isotopes. Through cell lysis, membranes were collected from the pooled samples from which proteins were prepared for MS. Upon digestion of proteins, the collected peptides were measured using SRM. In wildtype control cells all OST components had a normal level (100%) (Fig. 10C). Upon deletion of the Stt3 protein, the cells became LmSTT3D-dependent due to loss of activity of endogenous OST, and the amounts of Ost3p and Ost6p were greatly reduced showing destabilization of these proteins. These results are in accordance with previous studies showing that Stt3 deletion alters the amounts of Ost3p and Ost6p by inducing a faster degradation of these subunits 35. For all the stt3p point-mutants, we observed a different scenario. All subunits were expressed to a hundred percent relative to each other, but peculiarly, they were expressed in higher amounts relative to the wild-type. This implied an upregulation of OST subunit proteins upon mutation of residues involved in catalysis. These results showed that subunit proteins were present in stoichiometric amounts required for the formation of stable complexes, but that such perturbations induced an upregulation of OST proteins probably as either part of an unfolded protein response or to produce more complexes in an attempt to preserve enzyme activity. We concluded that mutations involved in either the coordination of the metal ion or interaction with the phosphate of the LLO substrate inactivate OST activity without destabilizing the complex. 54

66 Yeast stt3p point-mutations affect OST complex assembly but not stability of Stt3p Previously characterized stt3p mutants showed reduced OST activity, with hypoglycosylation and temperature sensitive phenotypes at 37 C 7,42. This reduced activity, for example, in the stt3-7 mutation (S552P) was attributed to complex destabilization resulting from the degradation of the mutated stt3 protein. We however found earlier that mutation of residues involved in catalysis did not destabilize stt3p itself, so we wondered if there were other stt3p mutations that did not destabilize Stt3p but affected the assembly and stability of the whole complex. We identified residue D71 and N539 located in the luminal domain of Stt3p. D71 is found on the same helix as the catalytic D47 amino acid and is conserved in prokaryotic and eukaryotic OSTs (Fig. S3). We mutated D71 to an alanine and analysed its effect on enzyme activity. D71A mutation retained some OST activity and cells grew on 5FOA (Fig. 11.1A). This mutation however reduced fitness and resulted in temperature sensitivity at 37 C. We further analysed the effect of D71 mutation on complex stability using SILAC pooling experiments. Our results showed reduced steady state levels of Ost3p and Ost6p when compared to the wild-type (Fig. 11.1C), implying that the complex was destabilized. This phenotype was similar to the phenotype observed in the stt3p deletion strain except for the fact that Stt3p itself remained stable. To confirm that the reduced levels of Ost3p and Ost6p were due to degradation, we conducted a pulse SILAC-SRM experiment and determined degradation rates for all subunit proteins. Proteins from membranes of wild-type yeast and D71A mutant strain were processed for mass spectrometry and quantified using SRM. It was observed that all subunits were stable in the wild-type strain. In contrast, Ost3p and Ost6p were degraded at a higher rate in the D71A strain when compared to the wild-type (Fig. 11.1D) while all other subunits remained stable. These results confirmed that D71A destabilized the OST complex resulting in the degradation of Ost3p and Ost6p. OST3 and OST6 deletions has not such a strong phenotype, suggestive of another role for this residue. On the other hand, N539 is one of the predicted potential N-glycosylation sites of Stt3p located on the C-terminal part of luminal domain. It is known that Stt3p is a 55

67 glycoprotein with three predicted sites, with the other two being N60 and N535. In contrast to N60 which is not conserved at all, N535 and N539 are conserved in all complex-forming OSTs but not in single subunit OSTs of lower eukaryotes (Fig. S3). Figure 11.1: Mutation D71A reduces OST activity through destabilization of complex (A) Yeast strains containing a single point-mutation on the stt3 protein were complemented by a URA3 plasmid encoding the Leishmania major STT3D protein. Serial dilutions of mutants were spotted on medium containing 5FOA and incubated at 23 C for several days to select for ura - cells. Mutations that inactivate the enzyme do not support growth of 5FOA. (B) Serial dilutions of the point mutants that grew on 5FOA were spotted on YPD medium to check for temperature sensitivity. (C) Yeast stt3 point-mutants were grown in light amino acid-containing medium and wild-type in heavy amino acid containing medium. MS samples were prepared from equal-weight-pooled samples and measured by SRM. OST subunit protein abundance relative to wild-type are shown for mutants. (D) Degradation rates of OST subunits in wild type and D71A mutant were calculated as described in above. Values below the line indicate a half-life less than 12hrs representing 8 cell divisions. Data shown represent 3 biological replicates. We mutated both N535 and N539 to alanine and investigated the effect on enzyme activity. Both mutations did not abolish but greatly reduced growth. These results where contradictory to previous research showing that mutations of these residues were lethal 43. This reduction was more severe in mutant N535A resulting in a temperature sensitive phenotype at 37 C (Fig 11.2A). To verify which of the sites was glycosylated, wild-type yeast cells were lysed and membranes were enriched for glycopeptides. The glycans were cleaved with Endoglycosidase H (Endo H) maintaining the first GlcNAc residue on the peptide. Glycopeptides were then analysed by LC-MS to identify sites that had been occupied. 56

68 Figure 12.2: Mutations D71A and N539A destabilize OST complex but not Stt3p. Serial dilutions of yeast strains containing stt3p point-mutations that supported growth on 5FOA were spotted on YPD medium (A) and incubated overnight at different temperatures to check for temperature sensitivity. (B) Yeast stt3p point mutants were grown in light amino acid-containing medium and wild-type in heavy amino acid containing medium. MS samples were prepared from equalweight-pooled samples and measured by SRM MS. OST sub-unit protein abundance relative to wildtype are shown for mutants. (C, upper panel) Wild-type yeasts grown in heavy medium were mixed in a one -to -one ratio with stt3p mutant grown in light medium. From the pooled samples, proteins were digested and the glycans cleaved with Endo H, leaving behind a peptide decorated with a single GlcNAc residue. Relative quantification of every glyco-site provided occupancy values for the mutant relative to the wild-type. (C, lower panel) Protein expression levels greater than 120% (above the dashed line) relative to wild-type indicated upregulation in expression. Data shown represents 3 biological replicates. The two sites are present on the same tryptic peptide and MS data on wild-type yeast identified a single glycan attached to the peptide but results were inconclusive as to which site was occupied. We then took advantage of the two mutations which would independently block a single glycosylation site. We identified a glycosylated peptide in mutant N535A mutant but not in the N539A mutant. These results indicated that N539 is the site glycosylated in wild-type yeast (Fig. S4) and that blocking this site did not 57

69 lead to glycosylation of the N535 site as previously reported by Li and colleagues 43. This was further confirmed by performing the same experiments in the presence of the complementing LmSTT3D protein. We showed that the Leishmania homologue LmSTT3D could also not glycosylate the N535 site (data not shown). In the next step, we analysed the effect of both mutations on OST complex stability. N539A mutation destabilized OST complex as shown by reduced levels of Ost3p and Ost6p within the complex when compared to wild-type (Fig. 12.2B). More so, the STT3 subunit was not destabilized by this mutation. Based on these results, we suggested that Stt3p glycosylation confers stability to the OST complex and removal of the glycan induces degradation of some subunit proteins. Intriguingly, N535A maintained a stable OST complex despite the more severe growth phenotype displayed. We then analysed the glycosylation efficiency of both mutants by SDS-PAGE and immunoblot (Fig. S1A). Results from CPY, Ost1p and Wbp1p glycosylation showed that both mutations lead to hypoglycosylation of proteins, but these results could not explain the differences in the growth phenotype observed. We further evaluated in a more systematic way the global glycosylation efficiency of both mutations. We conducted SILAC experiments, where wild-type cells grown in medium supplemented with heavy isotopes of arginine and lysine were pooled in a one-to-one ratio with N535A or N539A mutants grown in medium with light isotopes. Cells were lysed, and proteins were processed for MS measurements. Glycoproteins where digested enzymatically and glycans were cleaved with Endo H maintaining the first GlcNAc residue on the peptide. Peptides were quantified by PRM and glycosylation-site occupancy for each site was quantified in a mutant relative to wild-type. We observed that on a global scale, both mutants severely hypoglycosylate yeast proteins, but did so to a different extend (Fig. 12.2C). At the protein level, both mutations resulted in the upregulation of molecular chaperons and foldases such as Ero1p and Pdi1p, which are known to be involved in the unfolded protein response (UPR) and other proteins such as Ktr1p and Pmt2p, important for O-glycosylation of proteins. This upregulation was however more pronounced in mutant N535A with up to four folds' increase for some proteins, and barely evident for mutant N539A. Interestingly, there was also an upregulation of all OST proteins in N535A but not N539A (Fig. 12.2B and C). These results further emphasized the more severe effect of N535A as already observed in the temperature sensitive growth phenotype. Based on all these results, we concluded that mutations of 58

70 D71 and N539 reduced OST efficiency through destabilization of the complex, and not through direct influence on catalysis. N535A on the other hand greatly reduces enzyme activity but does not destabilize the OST complex. Yeast stt3p point-mutations severely reduced OST activity but do not affect complex stability Based on the presumption that residues that play an important role in determining catalytic activity are conserved, we identified two highly conserved residues; W75 and K586. W75 is located on the large luminal-oriented external loop one (EL 1) that connects the first two transmembrane helices in the N-terminus of Stt3p. K586, on the other hand, is located on the hydrophilic C-terminal domain of the Stt3p close to the well-studied peptide binding WWDYG motif. Mutation of both residues to alanine did not inactivate OST, but caused severe growth defects. W75A had the most severe phenotype exhibiting complete loss of activity at 37 C while K586 retained a very low level of activity at this elevated temperature (Fig. 13A). Next, we used SILAC pooling experiments as described above to determine the effect of these mutations on OST complex assembly or stability. Interestingly, mutating these residues had no effect on the incorporation of subunits into the complex and all proteins were present in stoichiometric amounts required for the formation of stable complexes (Fig. 13B). Considering the severity of the growth defect, we determined the effect of the mutant on enzyme activity by analysing glycosylation efficiency of yeast proteins on a broad scope using SDS-PAGE immunoblotting and SILAC-PRM as previously mentioned. Our results demonstrated that W75A and K586A both caused a systematic and severe hypoglycosylation of yeast proteins. W75A caused a more severe phenotype in which 64% of all sites analysed were glycosylated to <10% relative to wild-type compared to 61% of all sites in K586 glycosylated to the same extend. In addition, 42% of the glycosylation sites in W75 were not occupied at all as compared to only 36% in K586. Only a few sites including the Stt3p N539 site described above remained glycosylated to a 100%, indicative of the importance of the glycan on this site for enzyme activity. Quantification of protein amounts in the mutants showed an upregulation of proteins involved in the UPR (Ero1p, Pdi1p, and Lhs1p), O-glycosylation (Ktr1p and Pmt2p) and OST subunit proteins (Fig. 13B and C). 59

71 Figure 13: Mutations of stt3p residues do not affect complex stability but greatly reduce enzyme activity. Serial dilutions of yeast strains containing stt3p point-mutations that supported growth on 5FOA were spotted on YPD medium (A) and incubated overnight at different temperatures to check for temperature sensitivity. (B) Yeast stt3p point-mutants were grown in light amino acid-containing medium and wild type in heavy amino acid containing medium. MS samples were prepared from equal- weight-pooled samples and measured by SRM MS. OST subunit protein abundance relative to wild-type are shown for mutants. (C, upper panel) Wild-type yeasts grown in heavy medium was mixed in a one -to -one ratio with stt3p mutant grown in light medium. From the pooled samples, proteins were digested and the glycans cleaved with Endo H leaving behind a peptide decorated with a single GlcNAc residue. Relative quantification of every glyco-site provided occupancy values for the mutant relative to the wild-type. (C, lower panel) Protein expression levels were measured for yeast proteins including OST subunits. Data shown represent 3 biological replicates. 60

72 We also observed that some proteins, for example Crh2p and Ecm33p involved in cell wall biosynthesis or Fet3p and Fet5p involved in iron transport were present in much reduced amount (<60% relative to wild type) either as a result of down regulation of protein expression or degradation. These results from the quantification of glycosylation-site occupancy and protein amounts relative to wild-type explained the growth phenotype observed for these point-mutants. We concluded that W75A and N353A described above and K586A greatly reduced OST activity through a mechanism that neither involves direct catalysis nor complex assembly. 61

73 Discussion Stt3 is the most conserved of all OST subunits and its physiological importance is highlighted by two congenital diseases of glycosylation caused by mutations in this protein 44. Previous functional studies have identified and characterized Stt3p mutants based on the conservation of residues across different species or in genetic screens 42,43,45,46. Here, we describe the identification and characterization of novel stt3p mutants based on its predicted structure using a reverse genetics approach. We generated chromosomal mutations and used a combination of SILAC-SRM and PRM mass spectrometric analytical methods to study the effect of these mutations on enzyme activity and OST complex formation in vivo. This genetic approach allowed the study of the function of the Stt3p in a physiologically relevant context by maintaining a single but altered gene copy of it at its original locus. In addition, the whole experimental strategy had the advantage of allowing the quantification of many protein glycosylation sites at the same time, revolutionizing the previous growth and CPY methods to a broader systemic view of OST activity. The crystal structure of bacterial OST showed that the active site is comprised of three acidic residues; D56, D154, and E Similarly, in the context of a four-coordinated metal ion, we identify residues D47, D166, E350 and E168 as catalytic residues of yeast stt3p. D47 is located on the EL1 while D166 and E168 belong to the previously characterized DXD motif 15,45 located at the interface between the transmembrane and luminal domains. Residue E350, the corresponding residue of the PglB E319, was located on the flexible external loop 5 and has not previously been characterized in yeast. Mutations of all four residues to other amino acids completely inactivated OST without affecting complex assembly (Fig. 10), supporting their identity as critical residues involved in catalysis. In addition to the coordination of the metal ion, another vital interaction for the transfer reaction is that of OST to its LLO substrate. We identified arginine at position 404, as the OST residue that possibly interacts with the incoming LLO substrate. Mutation of this arginine 404 to an alanine or glutamate abolished enzyme activity whereas its replacement with a different positively charged residue lysine only reduced enzyme activity (Fig. S1B). This enzyme inactivation could therefore be attributed to removal of the positive side chain or introduction of a negative charge as the mutation R404K did not abolish enzyme activity; though it greatly reduced it. These results suggested that a positively charged residue is required 62

74 at position 404 of Stt3p for enzyme activity and that maximum activity is conferred by an arginine. The requirement of a positively charged residue at this position possibly ensures an interaction with the negatively charged phosphate group of the LLO, thereby confirming the location of the putative carbohydrate binding site. Since similar results have been obtained for homologues of these residues in archaea 19 and bacteria 18, our results further assert the hypothesis of a similar structural architecture for prokaryotic and eukaryotic OSTs. With respect to the reaction mechanism, the EL5 was characterized to play two essential roles in prokaryotic OSTs. Its C-terminal part pins a bound sequon to the periplasmic domain, providing the essential glutamate to the active site and correctly positions the Asn for glycosylation. The N-terminal part on the other hand, contains the tyrosine plug involved in the possible stacking of the LLO substrate to the catalytic site allowing the pyrophosphate moiety to interact with the conserve arginine and the divalent metal ion to increase the OST transfer rate 47. In contrast to the C-terminal part of the EL5 which is conserved in prokaryotes and eukaryotes, the N- terminal part harbouring this tyrosine plug is not similar to that of yeast and other eukaryotic OSTs. We therefore propose a mechanism for eukaryotes in which E350 of the EL5 participates in coordination of the metal ion and R404 interacts with the LLO substrate in similar ways as in the PglB but differing with respect to the stacking interaction. The absence of the tyrosine plug implied that the LLO stacking mechanism was different, probably explained by differences in the structure of eukaryotic and prokaryotic LLOs. Furthermore, since yeast Stt3p is part of an octameric complex, it is tempting to speculate that the function of the N-terminal part of the EL5 is being performed by another subunit. This however does not explain the absence of such an interaction in single subunit eukaryotic OSTs. It is known that Stt3p is a glycoprotein with three potential glycosylation sites; N60, N535 and N539. In accordance with previous reports 43, we confirmed that only one of the three potential sites, N539 is glycosylated in wild-type. However, we demonstrated that blocking site N539 did not result in the glycosylation of the neighbouring N535 site (Fig. S4) and that N535 and N539 mutations did not result in lethality as previously described 43. Perhaps the previous experimental set-up where Stt3p was analysed as a C-terminally Hemagglutinin-tagged protein on the prs314 plasmid was susceptible to the generation of such false phenotypes. Our results further emphases the need for proper analytical tools and reiterate the advantage of our experimental strategy. 63

75 Considering the bulky nature of the eukaryotic LLO, we imagine that spatial restriction possibly accounts for the inability of the N535 to be glycosylated. The high conservation of N535 and N539 in only complex-forming OSTs highlighted by the structural alignment was suggestive of a complex stability-related function. Indeed, the absence of the glycan resulted in destabilization of OST complex (Fig. 12.2B). N-linked glycans play an important role in proper folding and stability of proteins 4,12,48. Interestingly, the loss of the Stt3p glycan did not affect Stt3p stability, but rather that of the Ost3p and Ost6p proteins, which are found within the same sub-complex as Stt3p in OST. The decrease of ost3/ost6 proteins is probably due to a loss of interaction between these subunits and a misfolded Stt3 protein resulting from the absence of the glycan. Alternatively, it could be due to a loss of direct interaction between the proteins and the missing carbohydrate that partially mediate the interactions between the three proteins. In either case, Ost3/Ost6 does not properly integrate into the complex and are degraded. The stability of Stt3 protein in a complex-destabilizing scenario also observed in the D71A mutation (Fig. 12.1C) might suggest it stoichiometric location within the complex which prevents its degradation. The most striking mutants isolated in our study were those not involved in direct catalysis but which greatly affected glycosylation efficiency of OST without destabilizing the complex. Analysis of site occupancy for different substrate peptides in these mutant-strains relative to wild-type showed a systematic and severe hypoglycosylation of yeast proteins. We questioned what these mutants (W75A, N535A and K586A), which portrayed the most severe hypoglycosylation phenotype do? Several factors such as LLO structure, composition of the OST complex and the peptide sequence neighbouring the N-X-S/T sequon are known to influence the efficiency of N-linked glycosylation of proteins Eliminating complex composition as a possible factor, we regarded these mutants as being involved either in peptide or carbohydrate binding interactions. Residue K586 on the predicted structure was located close to the peptide binding motif WWDYG and is part of the previously described DK motif 15. Lysines are known to be important for structural integrity, usually involved in the formation of salt-bridges 52. In contrast to previous results that characterized K586 as a lethal mutation that 64

76 interacts with the pyrophosphate group of the LLO 15, we propose that lysine forms a salt-bridge with the negatively charged glutamate of the WWDYG motif stabilising it for proper peptide binding during N-linked glycosylation. Upon mutation, this saltbridge is abrogated resulting in a conformational change of the motif. This also explained the systemic nature of the hypoglycosylation phenotype observed. It is worth noting that despite the severe phenotype, K586A mutant was viable in contrast to the lethal phenotype previously described 15. We attributed this viability to either the effect of the highly activated unfolded protein response or the hypoglycosylation of only nonessential proteins. Along these lines, it is reasonable to presume that OST complex proteins either act as chaperons to each other or are simply involved in the UPR mechanism as seen by their upregulation. W75A, a novel stt3p mutant showed a more severe hypoglycosylation phenotype when compared to an alg9 deletion strain (Fig. S5) and exhibited more similarity to K586A mutant in a hierarchical cluster analysis (Fig. S6). Cluster analysis groups objects into groups, where group members share common properties making it easier to predict the properties of an object based on group membership. By using cluster analysis, we attempted to predict the unknown functions of our stt3p mutants based on the function of group members. Our results suggested that W75A did not affect LLO substrate interaction, or at least that such an interaction was not solely responsible for the observed phenotype. However, it's clustering together with K586A may be indicative of a peptide substrate interaction role. N535 on the other hand is located on a flexible loop very close to the catalytic site in the carbohydrate substrate-binding pocket. It probably plays a structural role ensuring a correct conformation for efficient glycosylation. Arguably, stable complexes do not necessarily translate to correctly assembled complexes. The possibility also exists that mutations that did not destabilized the OST complex could have affected its assembly (altering interactions between subunits but not enough to destabilize the whole complex), consequently reducing enzyme activity. Finally, we found out that even though the single subunit LmSTT3D protein could glycosylate yeast proteins, its activity was completely different from that of yeast stt3p, shown by the complete separation of the lethal D47A and stt3p deletion strains from 65

77 yeast OST mutants in our cluster analysis (Fig. S6). Additionally, we showed that the glycosylation efficiency of LmSTT3D did not change in the absence or presence of inactive endogenous complexes, indicating the lack of competition between the enzymes in which the inactive complex would sequester substrates away from the LmSTT3D (Fig. S2) This study has provided greater insight into the function of the eukaryotic OST by assigning and providing better explanations and functions to previously described mutants together with the identification and characterization of novel mutants. High resolution structures of the OST complex and/or Stt3p are required together with more experimental work to validate these findings. 66

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83 72

84 Chapter 2: Supplementary information 73

85 FigureS1: Mutations of stt3p residues affect the efficiency of glycosylation of yeast proteins. Equal amounts of proteins from yeast stt3p mutants grown in YPD medium and lysed with glass beats and detergent were analysed by immunoblot. All mutations resulted in hypoglycosylation of Ost1p, Wbp1p and CPY (A). Yeast stt3p point-mutant was grown in light amino acid-containing medium and wild-type in heavy amino acid containing-medium. MS samples were prepared for equal weight-pooledsamples and measured by SRM MS. OST subunit protein abundance relative to wild-type are shown for mutant (B, upper panel). Wild-type yeasts grown in heavy medium were mixed in a one-to-one ratio with stt3p mutant grown in light medium. From the pooled samples, proteins were digested and the glycans cleaved leaving behind a peptide decorated with a single GlcNAc residue. Relative quantification of every glyco-site provided occupancy values for the mutant relative to the wild-type (B, lower panel). Protein expression levels were measured for yeast proteins including OST subunits. R404K showed reduced glycosylation efficiency of OST. Data shown represent 3 biological replicates. 74

86 Figure S2: Mutations that inactivate OST do not sequester substrate in the presence of LmSTT3D. Yeast stt3p point-mutants were grown in light amino acid-containing medium and wild-type in heavy amino acid containing-medium. MS samples were prepared from equal-weight-pooled samples and measured by SRM MS. OST subunit protein abundance relative to wild-type are shown for mutants (A). Wild-type yeasts grown in heavy medium is mixed in a one-to-one ratio with stt3p mutant grown in light medium. From the pooled samples, proteins were digested and the glycans cleaved, leaving behind a peptide decorated with a single GlcNAc residue. Relative quantification of every glyco-site provided occupancy values for the mutant relative to the wild-type (C). Protein expression levels were measured for yeast proteins including OST subunits (D). All mutants analysed showed reduced glycosylation efficiency of OST. Data shown represent 3 biological replicates. 75

87 Figure S3: Alignment of protein sequences of OSTs from different organisms. Protein sequences from PglB and eukaryotic OSTs were aligned, and identical residues are displayed in black. Conserved residues that were mutated are depicted with red vertical boxes over the sequences. FigureS4: Yeast Stt3p is glycosylated at position N539 Membranes were collected from lysed cells and enriched for glycopeptides using ZIC HILIC column. Glycopeptides were treated with Endo H which cleaves off the glycan between the two core GlcNAc residues generating a peptide tagged with a single GlcNAc. Glycopeptides were analysed by Velos- Orbitrap mass spectrometer. The presence of the GlcNAc was used to identify a site that had been occupied by a glycan. In the N535A strain, MS/MS spectrum at m/z of peptide TTLVDANTWNNTHIAIVGK contained one GlcNaC residue. In mutant N539A, the peptide eluded at a different retention time and MS/MS data did not support the peptide sequence containing a GlcNAc molecule. 76

88 FigureS5. Deletion of alg 9 reduces OST activity and activates the UPR machinery. Wild-type yeasts grown in heavy medium was mixed in a one-to-one ratio with yeast cells deleted of the alg9 gene grown in light medium. From the pooled samples, proteins were digested and the glycans cleaved, leaving behind a peptide decorated with a single GlcNAc residue. Relative quantification of every glyco-site provided occupancy values for the mutant relative to the wild type (A). Protein expression levels were measured for yeast proteins including OST subunits (B). Data shown represent 3 biological replicates. 77

89 Missing value Figure S6: Glycosylation-site occupancy profile of yeast OST mutants. The colour scale represents glycosite occupancy for yeast stt3p mutants and alg9 deletion for 3 biological replicates. The similarity in the glycosylation profiles between mutants was calculated using the Euclidean distance and the dendogram was obtained by complete linkage clustering. 78

90 Table S1: Yeast strains in this study Name Parent strain Genotype Reference BY4742 BY4742 his3 1; lys2 0; ura3 0; leu2 0 EUROSCARF KP4 BY4742 arg4 0 his3 1; lys2 0; ura3 0; leu2 0 Kristina Poljak EN0 BY4742 Matα stt3:: K. lactis leu2; arg4 0; his3 ; lys2 0; ura3 0 This study EN1 BY4742 Matα stt3:: STT3 arg4 0; his3 1; lys2 0; ura3 0 This study EN2 BY4742 Matα stt3:: stt3n61a arg4 0; his3 1; lys2 0; ura3 0 This study EN3 BY4742 Matα stt3:: stt3nf50a arg4 0; his3 1; lys2 0; ura3 0 This study EN4 BY4742 Matα stt3:: stt3n51a arg4 0; his3 1; lys2 0; ura3 0 This study EN5 BY4742 Matα stt3:: stt3y52a arg4 0; his3 1; lys2 0; ura3 0 This study EN6 BY4742 Matα stt3:: stt3r53a arg4 0; his3 1; lys2 0; ura3 0 This study EN7 BY4742 Matα stt3:: stt3r32a arg4 0; his3 1; lys2 0; ura3 0 This study EN8 BY4742 Matα stt3:: stt3k586a arg4 0; his3 1; lys2 0; ura3 0 This study EN9 BY4742 Matα stt3:: stt3n535a arg4 0; his3 1; lys2 0; ura3 0 This study EN10 BY4742 Matα stt3:: stt3n539a arg4 0; his3 1; lys2 0; ura3 0 This study EN11 BY4742 Matα stt3:: stt3w75a arg4 0; his3 1; lys2 0; ura3 0 This study EN12 BY4742 Matα stt3:: stt3d71a arg4 0; his3 1; lys2 0; ura3 0 This study EN13 BY4742 Matα stt3:: stt3d71a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN14 BY4742 Matα stt3:: stt3y76a arg4 0; his3 1; lys2 0; ura3 0 This study EN16 BY4742 Matα stt3:: LEU2 arg4 0; his3 1; lys2 0; ura3 0 This study EN15 BY4742 Matα stt3:: stt3h351 arg4 0; his3 1; lys2 0; ura3 0 This study EN17 BY4742 Matα stt3:: stt3d47a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN18 BY4742 Matα stt3:: stt3d166a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN19 BY4742 Matα stt3:: stt3e168n arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN20 BY4742 Matα stt3:: stt3e350a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN21 BY4742 Matα stt3:: stt3e350q arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN22 BY4742 Matα stt3:: stt3r80a arg4 0; his3 1; lys2 0; ura3 0 This study EN23 BY4742 Matα stt3:: stt3i344a arg4 0; his3 1; lys2 0; ura3 0 This study EN24 BY4742 Matα stt3:: stt3ri345aa arg4 0; his3 1; lys2 0; ura3 0 This study EN25 BY4742 Matα stt3:: stt3r404a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN26 BY4742 Matα stt3:: stt3r404e arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN27 BY4742 Matα stt3:: stt3r404k arg4 0; his3 1; lys2 0; ura3 0 This study EN28 BY4742 Matα stt3:: stt3w325a arg4 0; his3 1; lys2 0; ura3 0 This study EN29 BY4742 Matα stt3:: stt3t326a arg4 0; his3 1; lys2 0; ura3 0 This study EN30 BY4742 Matα stt3:: stt3f329a arg4 0; his3 1; lys2 0; ura3 0 This study EN31 BY4742 Matα stt3:: stt3y330a arg4 0; his3 1; lys2 0; ura3 0 This study EN32 BY4742 Matα stt3:: stt3g209a arg4 0; his3 1; lys2 0; ura3 0 This study EN33 BY4742 Matα stt3:: stt3f213a arg4 0; his3 1; lys2 0; ura3 0 This study EN34 BY4742 Matα stt3:: stt3n61a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN35 BY4742 Matα stt3:: stt3f50a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN36 BY4742 Matα stt3:: stt3n51a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN37 BY4742 Matα stt3:: stt3y52a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN38 BY4742 Matα stt3:: stt3r53a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN39 BY4742 Matα stt3:: stt3r32a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN40 BY4742 Matα stt3:: stt3r404k arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN41 BY4742 Matα stt3:: stt3w75a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN42 BY4742 Matα stt3:: stt3w325a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN43 BY4742 Matα stt3:: stt3t326a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN44 BY4742 Matα stt3:: stt3f329a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN45 BY4742 Matα stt3:: stt3y330a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study 79

91 Table S1 continue Name Parent strain Genotype Reference EN46 BY4742 Matα stt3:: stt3k586a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN47 BY4742 Matα stt3:: stt3n535a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3 This study EN48 BY4742 Matα stt3:: stt3n539a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN49 BY4742 Matα stt3:: stt3y76a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN50 BY4742 Matα stt3:: stt3h351a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN51 BY4742 Matα stt3:: stt3r80a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN52 BY4742 Matα stt3:: stt31344a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN53 BY4742 Matα stt3:: stt31345a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN54 BY4742 Matα stt3:: stt3g209a arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN55 BY4742 Matα stt3:: stt3f213 arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN56 BY4742 Matα stt3:: stt arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN57 BY4742 Matα stt3:: stt arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN58 BY4742 Matα stt3:: stt arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN59 BY4742 Matα stt3:: stt arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study EN60 BY4742 Matα stt3:: stt arg4 0; his3 1; lys2 0; ura3 0;LmSTT3D This study Table S2 Plasmid name Purpose Reference prs313 Template for cloning of STT3 plasmid construct (Chee and Haase, 2012) prs313-stt3-kanmx Carries STT3-KanMX construct for mutagenesis this study pfa6a-kanmx Template carrying KanMX casette for cloning STT3 construct (Wach et al., 1994) pax309 Carries LmSTT3d-HA-cyc1 YEp352 plasmid under GPD promoter Aebi lab pug6 Carries loxp-kanmx-loxp cassette for arg4 knockout (Wach et al., 1994) pug73 Carries LEU2 marker gene from Klyveromces lactis used for STT3 knockout (Gueldener et al., 2002) psh47 URA3 plasmid Carrying Cre recombinase under Gal promoter (Güldener et al., 1996) 80

92 Chapter 2B: Further characterisation of S cerevisiae Stt3p 81

93 Results and discussion Importance of the luminal domain of Stt3p Stt3 is the most conserved of the OST subunits and all Stt3 proteins share a similar topology with a hydrophobic N-terminal part containing transmembrane helices and a large soluble C-terminal domain located on the luminal side of the ER in eukaryotes and periplasm in bacteria 1. The yeast Stt3p has 13 transmembrane helices spanning the ER membrane followed by a hydrophilic luminal-oriented C-terminal domain consisting of 255 amino acid residues (Fig. 1A). Because the luminal domain is known to habour the WWDYG peptide binding motif 2 and the phyrophosphate binding site described in chapter 2, we reasoned that it must be essential for glycosylation, such that its deletion would result in lethality. We deleted the entire luminal domain (stt ) in the presence of the complementing Leishmania Stt3D protein and analysed the effect on growth. By subjecting the cells to growth on medium containing 5FOA where survival depended on endogenous OST activity, we observed that the deletion abolished enzyme activity causing lethality (Fig 1B). Since the complete deletion was lethal, we further asked whether there was a certain length requirement for catalytic activity such that portions of the domain could be removed without affecting enzyme activity. To address this question, we additionally prepared luminal domain truncations generating strains with Stt3p having 50 (stt ), 100 (stt , 150 (stt ), and 200 (stt ) amino acids left at its luminal domain. Growth analysis of these strains revealed that enzyme activity was already abolished upon deletion of 50 amino acid residues from the luminal domain. We concluded that the entire luminal domain is essential for OST activity. To determine how the enzyme was inactivated, we examined the effect of these deletions on OST complex stability by monitoring steady state levels of OST proteins. Using SILAC pooling experiments as described in chapter 2, we observed that in contrast to the wild type control cells where all subunits are expressed at normal levels (100%), the amounts of Ost3p, Ost6p and Stt3p were greatly reduced in our mutants (Fig. 1D). We had previously shown that the reduced amounts of subunits are due to degradation of the proteins upon perturbation using SILAC pulse experiments. Immunoblot analysis with antibodies against Stt3p for all deletions showed its absence confirming that Stt3p was degraded (Fig. 1C). 82

94 Figure 1: Deletion or truncation of Stt3p luminal domain destabilises OST complex. (A) Proposed architecture of Stt3p showing the 13 transmembrane and a large luminal hydrophilic domain. (B) Yeast strains containing stt3p with shortened or no luminal domain were complemented by a URA3 plasmid encoding the Leishmania major STT3D protein. Serial dilutions of mutants were spotted on medium containing 5FOA and incubated at 23 C for several days to select for ura - cells. Mutations that inactivate the enzyme did not support growth of 5FOA. (C) Equal amounts of proteins from yeast stt3p mutants grown in YPD medium and lysed with glass beats and detergent were analysed by immunoblot. (D) Yeast stt3p domain deletion mutants were grown in light amino acid-containing medium and wild type in heavy amino acid containing medium. MS samples were prepared from equal-weight-pooled samples and measured by SRM MS. OST subunit-protein abundance relative to wild type are shown for mutants. Asterics indicate OST subunits that are degraded upon deletion or truncation of stt3p luminal domain. Data shown represent 3 biological replicates. There was a noticeable upregulation of some subunit proteins probably as part of an unfolded protein response to help the cells overcome the stress conferred by the 83

95 deletions. These results together led to the conclusion that deletion or truncations of the luminal domain of Stt3p results in increased degradation of Ost3p and Ost6p and Stt3p thereby destabilizing the whole complex, and that this destabilization is responsible for the lethal phenotype. In addition to containing catalytic motifs, we propose that the luminal domain of Stt3p also plays an important structural role that ensures that stable active complexes are formed. Analysis of the N-terminal part of Stt3p As mentioned above, the N-terminal of Stt3p contain 13 transmembrane helices connected by long and short loops, in view of a similar architecture with the bacterial and archaeal enzymes 3,4. We identified a highly conserved area on one of the two long loops, EL1 with residues F5o, N51, Y52, R53, Y76 and R80 (Fig. 2) clustering around the catalytic site of Stt3p. We imagined that due to their location and conservation, these residues would be important for catalysis. Along this line of thought, we speculated that the residues with aromatic site chains would be involved in possible stacking interactions with the LLO substrate to increase transfer efficiency during N- glycosylation. If that were true, mutations of these residues to non-aromatic amino acids should either abolish enzyme activity or cause severe growth defects. We mutated these residues to alanine and evaluated their effects on enzyme activity. Despite the high conservation of these residues, the mutants surprisingly did not portray any visible growth phenotypes. By using SILAC pooling experiments, we observed that upon mutation of the above-mentioned residues, OST complex stability was not affected. We however observed a slight upregulation of OST proteins in mutant N51A and R80A. This indicated that at least these two perturbations slightly affected OST activity because there was a slight induction of the UPR but this was not enough to reduce cell growth. It would be interesting to know to what extend OST activity has to be affected to have a growth or glycosylation phenotype. It is worth noting that the slight induction of the UPR could also have been from another effect not related to OST activity which we did not assay for. Perhaps combined and not single point mutations are required to decipher the function of this conserved region or it may simply not be important for OST activity. 84

96 Figure 2: Alignment of protein sequences of OSTs from different organisms. Protein sequences from PglB and eukaryotic OSTs were aligned and identical residues are displayed in black. Conserved residues that were mutated are depicted with red vertical boxes over the sequences. Figure 3: Mutations of conserved residues on EL1 do not affect enzyme activity and complex stability Serial dilutions of yeast strains containing stt3 point-mutations that supported growth on 5FOA were spotted on YPD medium and incubated overnight at different temperatures to check for temperature sensitivity. Except for the W75A mutant already described in chapter 2, all other mutants showed no 85

97 growth phenotype. (B) Yeast stt3p point-mutants were grown in a light amino acid-containing medium and wild type in a heavy amino acid containing medium. MS samples were prepared from equal weightpooled samples and measured by SRM MS. OST subunit protein abundance relative to wild type are shown for mutants. Mutations did not destabilise OST complex. Data shown represent 3 biological replicates. Analysis of the N-terminal portion of the EL5 The EL5 is the second-long loop connecting the 9 th and 10 th transmembrane helices at the N-terminal part of the yeast Stt3p. While the C-terminal part of the loop contains the catalytic E35o residue described before and is responsible for peptide binding, the N-terminal half contains the tyrosine plug responsible for stacking of the LLO to the catalytic site for efficient N-glycosylation at least in prokaryotes 5. This disorder section of the loop is very different between the bacterial PglB and yeast Stt3p. More so, it is not conserved between the yeast and other eukaryotic OSTs. Nonetheless, this region in the yeast Stt3p contained several aromatic amino acid residues; W325, F329 and Y330, which could potentially play the same stacking role as described for PglB (Fig. 2). For the PglB, mutations of the conserved tyrosine plug residues; Y288, F292,Y293 to alanine showed that, residue Y293 was responsible for the impaired OST activity upon mutation with almost a complete loss of OST activity 5. We expected a similar phenotype from the mutation with one or more of the above-mentioned amino acids. Therefore, we mutated W325, F329 and Y330 to alanine and analysed their effects on growth and complex stability. No growth defects were observed and all subunit proteins were expressed to amounts similar to those in the wild type control strain, indicating the formation of stable OST complexes. We concluded that these mutations have no effect on enzyme activity and thus where not involved in the catalytic reaction as hypothesised. 86

98 Figure 4: Mutations of aromatic residues on EL5 does not affect enzyme activity or complex stability. (A) Serial dilutions of yeast strains containing stt3 point-mutations that supported growth on 5FOA were spotted on YPD medium and incubated overnight at different temperatures to check for temperature sensitive growth. (B) Yeast stt3 point-mutants were grown in a light amino acid-containing medium and wild type in a heavy amino acid containing medium. MS samples were prepared from equal weight-pooled samples and measured by SRM MS. OST subunit protein abundance relative to wild type are shown for mutants. Data shown represent 3 biological replicates. Analysis of other conserved residues This last set of residues were chosen for mutagenesis on the basis of high conservation together with a possible interaction with the LLO substrate during N-glycosylation. Residues G209, F213, I344, I345 and H351 were all mutated to alanine and the effect on growth and OST complex stability were analysed using the methods previously described. Mutations G209A, F213A, I344A and I345A did not affect enzyme activity and growth. OST subunit proteins were expressed in these mutants to a similar amount as in the wild type control strain illustrating that subunit protein stability was unaffected by these mutations (Fig. 5B). 87

99 Figure5: Mutations of conserved residues do not affect enzyme activity. Serial dilutions of yeast strains containing stt3p point-mutations that supported growth on 5FOA were spotted on YPD medium and incubated overnight at different temperatures to check for temperature sensitivity (A). Yeast stt3p point mutants were grown in a light amino acid-containing medium and wild type in a heavy amino acid containing medium. MS samples were prepared for equal weight-pooled samples and measured by SRM MS. OST subunit protein abundance relative to wild type are shown for mutants (B). On the other hand, H351A reduced in vivo OST activity resulting in a temperature sensitive phenotype at 37 C (Fig. 5A). Interestingly, H351A did not destabilize the OST complex but induced an unfolded protein response as there was upregulation of some proteins when compared to the wild type. We concluded that H351A affected enzyme activity through a mechanism different from direct catalysis or complex assembly. 88