USING COMPENSATION-ATTENUATED GENETICS TO UNDERSTAND UNDERLYING NETWORKS GOVERNING CELLULAR ROBUSTNESS WANG SIHUI (B.Sc. (Hons)), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2013 Declaration I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. 22/11/13 ------------------------------------- Wang Sihui i Acknowledgements I would like to express my gratitude to my supervisor, Associate Professor Davis Ng T.W., for his guidance and advice throughout the course of this research project. I would also like to thank Dr. Guillaume Thibault for helpful discussion and his willingness to share his knowledge and expertise. Special thanks go to members of the Cell Stress and Homeostasis group, Dr. Rupali Prasad, Dr. Shinichi Kawaguchi, Mr Anthony Tran, Mr Xu Cheng Chao, Ms Liu Ying and Ms Nassira Bedford, for their assistance these years in one way or another. I am grateful for the scholarship awarded by NUS Graduate School for Integrative Sciences and Engineering, without which this journey would not have been possible. I would also like to extend my thanks to the academic and technical staff at Temasek Life Sciences Laboratory for their invaluable help in my research. ii Table of Contents Declaration i Acknowledgements ii Abstract . vi List of Tables vii List of Figures viii List of Abbreviations . xi Chapter 1.Introduction 1.1.The secretory pathway in eukaryotes 1.1.1. Protein folding in the secretory pathway 1.1.2. Protein quality control . 16 1.2. The unfolded protein response 19 1.2.1. Sensing ER stress 21 1.2.2. UPR activation and regulation . 25 1.3. The UPR compensatory mechanism masks the phenotype of a loss in gene function 46 1.4. Using genetic screening as a means to dissect molecular pathways . 48 1.5. Thesis Rationale 51 Chapter 2. Materials and Methods 52 2.1. Yeast Strains, Media, and Cell Culture 52 2.1.1. Yeast strains . 52 2.1.2. Cell culture and media . 56 2.2. General molecular and biochemical techniques 58 2.2.1. Plasmids . 58 2.2.2. Primers used in this study 61 2.2.3. Reagents and Antibodies . 62 2.2.4. Cell Labeling and Immunoprecipitation (Pulse chase analysis) . 62 2.2.5. Growth Assay (Spotting) 63 2.2.6. Assessment of CPY folding using MalPEG conjugation to cysteine sulfhydryl groups 64 2.2.7. Western Analysis 64 2.2.8. Quantitative PCR 65 2.2.9. DNA Microarray . 66 2.3. Synthetic Lethality Screen . 67 2.3.1. UV Mutagenesis . 67 iii 2.3.2. Determining kill rate 67 2.3.3. Screening for temperature-sensitive synthetic lethal mutants . 68 2.3.4. Identifying Recessive Mutants . 69 2.3.5. Cloning and Sequencing Temperature-sensitive Mutants . 69 Chapter 3. Genetic screening for temperature-sensitive mutants displaying synthetic lethality with an ire1 null mutation . 73 3.1. Introduction 73 3.2. Genetic screening . 73 3.2.1. Screening by colony colour phenotype 74 3.2.2. Screening by counter-selection using 5-fluoroorotic acid (5-FOA) . 77 3.2.3. Screening by temperature sensitivity 79 3.3. Cloning by complementation 81 3.4. Secondary screen for biosynthetic and ERAD mutants 82 3.5. Results of genetic screens . 82 3.5.1. Summary of colony colour assay . 83 3.5.2. Summary of 5-FOA screen . 86 3.5.3. Summary of TS screen 88 3.6. Discussion 90 Chapter 4. The UPR buffers against a lethal pdi1 dysfunction 92 4.1. Introduction 92 4.2. Generating the pdi1-2 mutant in the W303 background . 93 4.3. Characterization of the pdi1-2 mutant . 96 4.3.1. pdi1-2Δire1 displays conditional lethality and retains endogenous proteins in the ER . 96 4.3.2. Pdi1pts is stable at the restrictive temperature 99 4.3.3. ER-retention of endogenous proteins is due to misfolding and not a general trafficking defect 100 4.3.4. Effect of pdi1 mutation on ERAD of misfolded substrates 102 4.4. UPR induction via Ire1p/Hac1p is necessary for viability of pdi1-2 106 4.5. Oxidation is not defective in pdi1-2 110 4.6. Other members of the PDI family are dispensable for pdi1-2 survival with UPR induction 113 4.7. High-copy suppressor screen identified NOP56 as a suppressor . 116 4.8. Microarray analysis 120 4.9. Verification of microarray data by quantitative PCR 126 iv 4.10. Expression of KAR2 suppresses defects in pdi1-2Δire1 128 4.11. Co-expression of KAR2 interacting factors has no additive effect over KAR2 expression alone 131 4.12. The Hsp70-like Lhs1p alone does not compensate for defects in pdi1-2Δire1 135 4.13. KAR2 and pdi1ts work synergistically in pdi1-2Δire1 . 138 4.14. Characterizing the functional interaction between KAR2 and pdi1ts 141 4.15. Discussion . 144 Chapter 5. Characterization of other mutants from the screen 152 5.1. Secondary screen for protein biogenesis defect 152 5.2. Secondary screen for ERAD defect . 159 5.3. tssl36 is a tip20 mutant . 163 5.4. tssl30 causes enhanced ERAD of CPY* 167 5.5. Discussion 169 Chapter 6. Conclusion and future direction . 171 7. References . 173 v Abstract The unfolded protein response (UPR) is a homeostatic mechanism in cells which is activated in response to accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER). The Ire1/Hac1 signaling pathway relays the UPR signal and activates a transcriptional programme which helps restore equilibrium in the ER by alleviating ER stress. Using compensation-attenuated genetics, a novel allele of protein disulfide isomerase (PDI), pdi1-2, was isolated. Pdi1p is an essential protein in Saccharomyces cerevisiae involved in the catalytic oxidation, reduction and isomerization of disulfide bonds in secretory and membrane proteins. pdi1-2 is inviable in the absence of the UPR, but UPR activation suppressed lethality and compensated for defects in the biogenesis of endogenous proteins, CPY and Gas1p. Microarray analysis suggested that the UPR is modulated over time and shows plasticity in its output in response to different types of stress. Surprisingly, PDI family members that are UPR target genes were dispensable for suppression of lethality in pdi1-2, suggesting they are not functionally interchangeable. pdi1-2 is oxidationcompetent, suggesting that the CPY folding defect may be due to a defect in its chaperone function. Upregulation of the Hsp70 chaperone Kar2p and its Hsp40 cofactors by the UPR helped buffer the lethal pdi1 dysfunction. Interestingly, coexpression of KAR2 and pdi1ts synergistically restored cell viability and CPY maturation to a level comparable to the UPR. It is likely that KAR2 specifically compensates for the chaperone defect in pdi1-2 during protein folding. This suggests that different chaperone networks in the ER can buffer one another during ER stress, and may work in synergy to contribute to cellular robustness. vi List of Tables Table 1. List of UPR target genes and their functional categories 28 Table 2. List of genes synthetic lethal with Δire1/Δhac1 based on SGA analysis 47 Table 3. Yeast strains used in this study 52 Table 4. Components of yeast culture media 57 Table 5. Plasmids used in this study 58 Table 6. List of primers used in this study . 61 Table 7. List of high-copy suppressor plasmids isolated and the genes encoded . 116 Table 8. List of genes encoded by the complementing plasmid . 165 vii List of Figures Figure 1. Overview of protein folding in the ER and the chaperones involved . Figure 2. Co-translational and post-translational translocation Figure 3. Domain organization of Hsp70 . Figure 4. Domain organization of Hsp40 . Figure 5. Substrate-binding cycle of Kar2p 11 Figure 6. Domain organization of PDI family members . 15 Figure 7. The Hrd1 and Doa10 complexes involved in ERAD . 18 Figure 8. The UPR signaling pathway in S.cerevisiae . 29 Figure 9. The three branches of the UPR in higher eukaryotes . 35 Figure 10. Diagrammatic representation of the ER stress response in higher eukaryotes . 41 Figure 11. Schematic representation of the BCL2 family of proteins under resting conditions and during ER stress . 44 Figure 12. Steps in the secretory pathway defined by temperature-sensitive yeast sec mutants deficient in protein secretion 49 Figure 13. Diagrammatic representation of the yeast adenine biosynthesis pathway 74 Figure 14. Primary genetic screen using colony colour phenotype 76 Figure 15. Primary genetic screen by counter-selection using 5-FOA . 78 Figure 16. Primary genetic screen by temperature sensitivity 80 Figure 17. Workflow of genetic screen using colony colour and the number of mutants obtained at each step 84 Figure 18. Workflow of genetic screen using 5-FOA and the number of mutants obtained at each step 87 Figure 19. Workflow of genetic screen using temperature sensitivity and the number of mutants obtained at each step 89 Figure 20. pdi1-2 contains a L476S point mutation in the a' domain of PDI1 . 92 Figure 21. Integrating the ts allele into the W303 genome 95 Figure 22. pdi1-2 is inviable at the restrictive temperature in the absence of the UPR 96 viii Figure 23. The ER forms of CPY and Gas1p accumulate in pdi1-2Δire1 at the restrictive temperature 98 Figure 24. Pdi1pts is stable at the restrictive temperature 99 Figure 25. The retention of CPY and Gas1p in the ER is not due to a general ER-golgi transport defect . 100 Figure 26. CPY is misfolded in pdi1-2 Δire1 at the restrictive temperature 102 Figure 27. CPY* is stabilized in the pdi1 mutant and the UPR does not fully compensate for this defect 103 Figure 28. ERAD of PrA* is affected in pdi1-2Δire1 . 104 Figure 29. ERAD of a non-glycosylated substrate is similarly affected in pdi1-2Δire1 105 Figure 30. The UPR is induced in pdi1-2 at the restrictive temperature . 106 Figure 31. Viability is mediated by Ire1p/Hac1p signaling branch 107 Figure 32. UPR activation fixes the defect in CPY maturation . 108 Figure 33. Deletion of the lumenal domain of IRE1 abolished suppression of lethality 109 Figure 34. Oxidation is not defective in pdi1-2Δire1 . 111 Figure 35. Addition of diamide did not improve oxidative protein folding . 112 Figure 36. Deletion of PDI family members has no effect on viability when the UPR is activated. . 114 Figure 37. The UPR sufficiently compensates for the defect in CPY processing in the absence of PDI family members. . 115 Figure 38. Isolates from high-copy suppressor screen 118 Figure 39. High-copy plasmids HC5 and HC7 partially suppressed cell lethality . 119 Figure 40. UPR target genes are differentially induced in pdi1-2, compared to WT 123 Figure 41. Microarray analysis identified UPR genes differentially upregulated in pdi1-2 at various time points after shifting to the restrictive temperature 125 Figure 42. Correlation between qPCR data with microarray data 127 Figure 43. Expression of KAR2 suppresses lethality 129 Figure 44. KAR2 improved CPY maturation in vivo 130 ix Figure 58. UPR activation inhibits enhanced degradation of CPY* WT, Δire1, tssl30Δire1, and tssl30 were pre-incubated at 37°C for 15 min, pulse labeled for 10 with [35S] methionine/cysteine, and chased for 60 min. Immunoprecipitation of CPY* was performed using anti-HA monoclonal antibody and normalized by total TCA precipitable counts. Proteins were separated by SDSPAGE, visualized by autoradiography, and quantified by phosphorimager analysis. Results are depicted in the graph above. Preliminary analysis suggested that the enhanced degradation is corrected by the UPR. When IRE1 was expressed, CPY* degradation proceeded at WT rate (Fig. 58). Since the mutant is synthetic lethal with Δire1, it suggests that the mutation causing enhanced degradation is detrimental to the cell. This could be because of indiscriminate degradation of all proteins, not just misfolded ones, resulting in the disruption of protein homeostasis. It would be interesting to identify the mutated gene and study its role in regulation of protein degradation, but unfortunately I was unable to clone this gene due to the extremely low transformation efficiency of this mutant. 168 5.5. Discussion Secondary screening for desired phenotypes is necessary for identifying mutants of interest. In this study, I screened the tssl mutants for defects in bioprocessing of CPY and Gas1p, as well as ERAD of CPY*. I identified two mutants that showed defects in protein folding/trafficking. The first mutant, tssl3, was later identified as pdi1-2 and described in detail in Chapter 4; the second mutant, tssl36, is a tip20 mutant and also has defects in ERAD. In addition, three other ERAD mutants were identified, two of which stabilized CPY*. Of interest is tssl30, which showed enhanced degradation of CPY*. It is unclear why the tip20 mutant (tssl36) has defects in both protein folding/trafficking, and ERAD, since it is known to be involved in retrograde transport. Perhaps it has an undiscovered function in anterograde transport, or the defects could be indirect effects arising from the disruption to normal recycling of factors that are involved in these processes. Initial characterization of tssl30 surprisingly showed enhanced degradation of the model ERAD substrate, CPY*. This could be due to the loss of quality control mechanisms governing degradation of misfolded proteins, or just proteins in general. Such a defect could cause a loss of protein homeostasis in the cell, which could lead to cell death. The role of the UPR in ensuring survival of these mutants needs to be further characterized, which would provide greater insights into the functions of these genes in protein trafficking and protein degradation. Unexpectedly, the number of colonies that needed to be screened to identify mutants of interest was much greater than initially thought. This is due to the high 169 proportion of false positives as well as the rarity of temperature-sensitive synthetic lethal mutants. As such, a more extensive screen is required if a broader range of mutants is to be identified. 170 Chapter 6. Conclusion and future direction The UPR provides an important buffer against dysregulation of protein homeostasis in the secretory pathway. In S.cerevisiae, the UPR is non-essential during non-stress conditions, but studies identifying the broad range of genes displaying synthetic lethality with the UPR underscore the importance of this stress response when cells undergo internal and external perturbations. In chapter and chapter of this thesis, I described the genetic system that was set up to allow us to study loss of gene functions in the absence of compensatory mechanisms of the UPR, as well as to examine how the UPR buffers against such physiologic stress. Chapter focuses on one of the mutants identified from the screen, pdi1-2, which exhibited a mild ERAD defect with the UPR on but was completely inviable in its absence. Analysis of this mutant suggested that even though there is only one UPR transducer in the budding yeast, the UPR displays plasticity and modifies its output according to the needs of the cell. As far as we know, Hac1p is the sole transcription factor activating the UPR target genes. How the cell regulates transcription of a specific subset of UPR genes when Hac1p is expressed remains an open question. One way we can study this is to examine the gene promoters that are bound by Hac1p over time using chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-SEQ). This will give us an idea whether Hac1p remains bound to all UPR target gene promoters over time, or whether its binding varies with time. Constant binding would signal the action of other repressors and additional signaling mechanisms that modifies the UPR output. 171 Interestingly, analysis of pdi1-2 revealed the cooperation between Kar2p and Pdi1p in protein folding. It is not known if this synergistic action occurs under normal condition, or is simply the result of buffering between different chaperone networks carrying out the same function of protein folding. 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Proc Natl Acad Sci U S A 103 (39), 14343-8. 183 [...]... identified in a study to find genes which complemented a karyogamy mutant (Rose et al 1989) Unexpectedly, this gene was found to be identical to the one cloned by a separate group trying to isolate the yeast homolog of BiP - a mammalian ER Hsp70 believed to help in the folding of membrane and secretory proteins (Normington et al 1989) KAR2 encodes a protein that is 67% identical to that of mouse BiP,... together to perform the important task of producing soluble proteins that are secreted and allow communication or interaction with the external milieu The secretory pathway consists of the rough endoplasmic reticulum (ER), ER exit sites, the ER to golgi intermediate compartment, the golgi complex and the subsequent transport of secretory vesicles The pathway is modulated by intracellular and extracellular... aims to increase ER folding capacity by ER expansion, increasing the number of chaperones and folding factors, increasing degradation of 20 misfolded proteins, and decreasing protein load through translation attenuation When ER homeostasis fails to be restored, apoptosis is initiated 1.2.1 Sensing ER stress Since folding of secretory and transmembrane proteins occur primarily in the ER, perturbations to. .. binds ER targeting sequences on nascent polypeptides, and localizes the ribosome complex to the ER via binding to the SRP receptor The nascent polypeptide is transported across the Sec61 channel while being translated In the post-translational mode, translated polypeptides in the cytosol are targeted to and imported into the ER via the TRC40 (mammalian) or GET (yeast) pathway As nascent polypeptide chains... 1.Introduction 1.1.The secretory pathway in eukaryotes As organisms evolve from prokaryotes to eukaryotes, there is increasing complexity in cellular layout, structure, and function One of the hallmarks of eukaryotes is the compartmentalization of the cell into distinct subcellular organelles, each with its own tailor-made environment that has been optimized for its specific function The secretory pathway in eukaryotes... processing of secretory and transmembrane proteins that pass through the secretory pathway It plays a pivotal role in ensuring that proteins fold into their native structures, and that unfolded/ misfolded proteins are recognized, retained, and targeted for degradation by quality control machineries To ensure that misfolded and unfolded proteins do not accumulate and lead to cell toxicity, the ER regulates... secretory capacity accordingly to deal with the demands of cell growth, survival and homeostasis (Farhan and Rabouille 2011) 1.1.1 Protein folding in the secretory pathway The ER is the main site where folding and processing of secretory and membrane proteins take place It has been estimated that a third of cellular proteins pass through the ER As such, the ER can be regarded as the protein folding factory... stress conditions as well, such as exposure to heavy metals and cytotoxic chemicals (NeuhausSteinmetz and Rensing 1997), oxidative insults, ischaemia/reperfusion and hemorrhagic shock (De Maio 1999) Members of the HSPs are classified according to their molecular weight In the ER of the budding yeast, two main groups of HSPs - Hsp70 and Hsp40, are present to maintain protein homeostasis Each member... glycoproteins in the ER to ensure their proper folding (Ellgaard et al 1999), and its yeast homolog, Cne1p, is believed to be involved in the folding of glycoproteins and their quality control (Parlati et al 1995) Cne1p possesses a conserved lectin domain which has been shown to bind monoglucosylated oligosaccharide, and a P- (proline-rich) domain that was shown to be required for Cne1p's ability to suppress aggregation... and that mammalian Ire1 similarly forms oligomers (Li et al 2010) Taken together, studies to date suggest that in yeast, ER stress is sensed by the direct binding of accumulated unfolded proteins to the cLD of Ire1p and promotes clustering of Ire1p Kar2p plays a modulatory role in this process by tuning the extent of UPR activation to be on par with the severity of the ER stress 1.2.1.2 Ire1, PERK, and . USING COMPENSATION -ATTENUATED GENETICS TO UNDERSTAND UNDERLYING NETWORKS GOVERNING CELLULAR ROBUSTNESS WANG SIHUI (B.Sc. (Hons)),. This suggests that different chaperone networks in the ER can buffer one another during ER stress, and may work in synergy to contribute to cellular robustness. vii List of Tables Table. were occurring (Shamaei-Tousi et al. 2007). HSPs were later found to be induced under other types of stress conditions as well, such as exposure to heavy metals and cytotoxic chemicals (Neuhaus- Steinmetz