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Discovery and Characterization of Prions in Saccharomyces Cerevisiae Discovery and Characterization of Prions in Saccharomyces cerevisiae by Randal A. Halfmann B.S. Genetics Texas A&M University, 2004 Submitted to the Department of Biology in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN BIOLOGY at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY FEBRUARY 2011 © 2011 Randal A. Halfmann. All rights reserved. The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part in any medium now known or hereafter created Signature of Author:_________________________________________________________________________________ Department of Biology December 3, 2010 Certified by:___________________________________________________________________________________________ Susan L. Lindquist Professor of Biology Thesis Supervisor Accepted by:__________________________________________________________________________________________ Stephen P. Bell Professor of Biology Co-Chair, Biology Graduate Committee 1 2 Discovery and Characterization of Prions in Saccharomyces cerevisiae by Randal A. Halfmann Submitted to the Department of Biology in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Biology ABSTRACT Some protein aggregates can perpetuate themselves in a self-templating protein-misfolding reaction. These aggregates, or prions, are the infectious agents behind diseases like Kuru and mad- cow disease. In yeast, however, prions act as epigenetic elements that confer heritable alternative phenotypes. Prion-forming proteins create bistable molecular systems whose semi-stochastic switching between functional states increases phenotypic diversity within cell populations. My thesis work explores the idea that rather than being detrimental, prions may commonly act to their host’s advantage. To broaden the known range of prion phenomena in S. cerevisiae, I, together with a postdoctoral fellow in our lab, systematically surveyed the yeast proteome for prion-forming proteins. Using a combination of computational, cell biological, and biochemical approaches, we ultimately identified 18 novel prion domains capable of driving phenotypic switching, and an additional 6 domains that were highly positive for prion-like aggregation in other assays. These results establish the critical importance of intrinsic amyloid-forming tendencies for prion behavior by Q/N-rich proteins. We further confirmed that one of these proteins, the transcription factor Mot3, forms a novel prion in its endogenous context. An analysis of these findings revealed a strong and unexpected amino acid bias in prionogenic proteins: prions were strongly enriched for asparagine (N), but not the chemically related amino acid glutamine (Q). We validated this finding using molecular simulations and experimental analyses of Q-to-N and N-to-Q variants of prion domains. N-rich sequences had an intrinsic tendency to both nucleate and propagate amyloid conformers. Q-rich proteins tended instead to make structurally non-constrained interactions leading to proteotoxic soluble and non- amyloid aggregated conformers. The appendices include works in progress. Each explores a different aspect of prion biology. Appendix A confirms a theoretical prediction that prions, if functional, should preferentially regulate certain rapidly evolving genes. I demonstrate with the newly discovered prion protein, Mot3, that prions accelerate the appearance of new phenotypes in important traits like mating behavior and cell-adhesion. I further identify naturally occurring prion states of Mot3 and other prion proteins in wild yeast isolates, and show that elimination of these prions has strong phenotypic effects in these strains. Appendix B, work done in collaboration with another lab, establishes that Nup100, a GLFG nucleoporin, is a prion. The conformational flexibility of GLFG nucleoporins is critical for the function of the nuclear pore complex, a molecular sieve that regulates all macromolecular transport between the nucleus and cytoplasm. Thesis supervisor: Susan Lindquist Title: Professor of Biology 3 4 Acknowledgements I will fondly remember my time here at MIT, and the people who helped me make the most of it. I am forever indebted to my advisor, Susan Lindquist. Quite simply, none of my work would have been possible without her guidance, enthusiasm and support. I hope that at least a fraction of the lessons I’ve learned with each trip to her office, whether to talk science or rewrite an abstract for the 42nd time, stay with me as I leave the lab. I will be a much better scientist for it. I want to thank my thesis committee members, Thomas Schwartz and Jonathan King, for sticking with me from beginning to end. My other interactions with Jon, first as his student and later as his teaching assistant, added valuable perspective to my research-driven graduate education. Teaching, as he explained, ensures that the next generation can pick up where we leave off. Finally, I thank Leona Samson and Eugene Shakhnovich for serving on my defense committee. The Lindquist lab has been a very supportive environment full of genuinely friendly and approachable people. Their extraordinary collective expertise, and willingness to share it with me, ensured that I never had to go far to find someone who could help. I want to especially thank Brooke Bevis for doing her job so well. She kept everything, and every one, running smoothly. I could always count on her insightful psychoanalyses to make sense of the few times when things did not go smoothly. I thank my baymate Ben Vincent for his quick and entertaining wit. I thank Kent Matlack for saving me the hassle of having to use a real thesaurus, and for showing me from time to time how to find beauty in the most unexpected places. I thank Dubi Azubuine for a constant supply of media no matter how frequent or unusual my requests. I am very fortunate to have had many talented collaborators both inside and outside the lab. Most importantly, I have to thank Simon Alberti. He has been a great friend and a valuable intellectual resource. I also thank my first postdoc mentor, Pete Tessier, for his limitless patience during my early and hectic days in the lab. Thanks to Oliver King, Charlie O’Donnell, and Alex Lancaster for doing insightful and magical things with computers. Similarly, thanks to Rohit Pappu and Nick Lyle for lending their expertise with molecular simulations. Finally, thanks to Jessica Wright and Michael Rexach for their contributions to the never-ending Nup100 saga. I want to thank the people who gave me my start. First, my parents for constant encouragement and for providing a sounding board no matter how bored I know they must have been. The lessons they’ve taught me from an early age, like the universal value of hard work, will always serve me well. I also thank my undergraduate advisor, David Stelly, who took me under his wing to get me started in research. Finally, I can’t imagine where I would be without Megan. Her constant love and companionship reminds me every day what is truly important. 5 6 Table of Contents Abstract 3 Acknowledgements 5 Table of Contents 7 Chapter One: Prions, Protein Homeostasis, and Phenotypic Diversity 9 Chapter Two: A Systematic Survey Identifies Prions and Illuminates 29 Sequence Features of Prionogenic Proteins Abstract 30 Introduction 30 Results 32 Discussion 40 Experimental Procedures 42 References 48 Figures 55 Chapter Three: Opposing Effects of Glutamine and Asparagine Dictate 83 Prion Formation by Intrinsically Disordered Proteins Summary 84 Introduction 84 Results 86 Discussion 92 Experimental Procedures 95 Supplemental Results and Discussion 100 References 102 Figures 107 Chapter Four: Epigenetics in the Extreme: Prions and the Inheritance 125 of Environmentally Acquired Traits Appendix A: Evolutionary Capacitance by Yeast Prions 135 Abstract 136 Introduction 136 Results 138 Discussion 142 Materials and Methods 144 References 145 Figures 147 Appendix B: Prion Formation by the GLFG Nucleoporin, Nup100 155 Abstract 156 Introduction 156 Results 158 Discussion 162 Materials and Methods 163 References 166 Figures 178 Appendix C: Specific Author Contributions 185 Appendix D: Curriculum Vitae 186 7 8 Chapter One Prions, Protein Homeostasis, and Phenotypic Diversity This chapter was published previously: Halfmann, R., Alberti, S., and Lindquist, S. (2010). Trends in Cell Biology 20, 125-33 9 Introduction Prions are self-replicating protein entities that underlie the spread of a mammalian neurodegenerative disease, variously known as Kuru, scrapie, and bovine spongiform encephalopathy, in humans, sheep and cows, respectively (Aguzzi et al., 2008). However, most prions have been discovered in lower organisms and in particular, the yeast Saccharomyces cerevisiae. Despite assertions that these prions, too, are diseases (Wickner et al., 2007a) (Box 1), many lines of evidence suggest that these mysterious elements are generally benign and, in fact, in some cases beneficial. In fungi, prions act as epigenetic elements that increase phenotypic diversity in a heritable way and can also increase survival in diverse environmental conditions (True and Lindquist, 2000; True et al., 2004; Shorter and Lindquist, 2005; Alberti et al., 2009). In higher organisms, prions may even be a mechanism to maintain long-term physiological states, as suggested for the Aplysia californica (sea slug) neuronal isoform of CPEB, cytoplasmic polyadenylation element binding protein. The
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