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Copyright by Jeremy Daniel O'connell 2012 Copyright by Jeremy Daniel O’Connell 2012 The Dissertation Committee for Jeremy Daniel O’Connell Certifies that this is the approved version of the following dissertation: Systemic Protein Aggregation in Stress and Aging Restructures Cytoplasmic Architecture Committee: Edward Marcotte, Supervisor Dean Appling Andrew Ellington Makkuni Jayaram Scott Stevens Systemic Protein Aggregation in Stress and Aging Restructures Cytoplasmic Architecture by Jeremy Daniel O’Connell, B.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin September 2012 Dedication Cytisus laburnum, simul vincet omnem To my dad and mom who encouraged and enabled my education with countless sacrifices, I promised this graduation would be the one we would attend, and I am truly sorry I was not swift enough to make that possible. Acknowledgements Foremost, I thank my advisor Edward Marcotte, for not just a second lease on a life in science but one in an amazing lab environment. His intellectual rigor, enduring patience, amazing work ethic, and enthusiasm for discovery were an inspiration. I thank my collaborators in this project: Gwen Stovall, Alice Zhao, Gabe Wu, and Mark Tsechansky for their comradery and support on this great adventure. I thank the talented undergraduates: Maguerite West-Driga, Ariel Royall, and Tyler McDonald who stuck with me. Each of you will soon be a better scientist than I ever will, and I hope you enjoyed and learned from our research together nearly as much as I did. Finally, I thank all the Marcotte lab members who moiled in the mines of systems biology with me. Many were the hours they sacrificed from their own projects to teach me, for that I am truly grateful. Thank you to Mark Carlson for teaching me cell culture techniques. Thank you to Traver Hart for introducing me to microscopy. Thank you to Kris McGary for the many discussions on the use of inductive and deductive reasoning in science (and a great many other fascinating topics). Thank you to Daniel Boutz for teaching me mass spectrometry. Thank you to Zhihua Li for teaching me the wonderful model system that is brewer’s yeast. There was not a day in the Marcotte lab when I was not happy to be there. v Systemic Protein Aggregation in Stress and Aging Restructures Cytoplasmic Architecture Jeremy Daniel O’Connell, Ph.D. The University of Texas at Austin, 2012 Supervisor: Edward M. Marcotte A common maxim of protein biochemistry states, “structure is function.” This is generally just as true for an individual polypeptide chains as for multi-protein complexes. The advent of yeast tagged-protein libraries has allowed systematic screening of a protein’s local interaction partners as well as a roughly mapping its cellular location. Recently our group and others discovered hundreds proteins forming new structures in stationary phase yeast cells using the yeast GFP-tag library. That equates to well over a quarter of normally diffuse cytoplasmic proteins assembled into discrete structures that appear as foci or fibers, all of unknown function. This study provides evidence that many of these foci are formed by protein aggregation- that contrary the maxim, structure can be dysfunction. Furthermore, this study uses yeast to demonstrate the generality of cytoplasmic protein aggregation in response to a variety of stresses, provides evidence that increasing aggregation of particular cytoplasmic proteins correlates with aging even across organisms, and proposes a theoretical framework for how cellular energy levels affect protein aggregation propensity. vi Table of Contents List of Tables ...........................................................................................................x List of Figures ........................................................................................................ xi List of Figures ........................................................................................................ xi Chapter 1: Introduction ............................................................................................1 Types of intracellular bodies ...........................................................................2 Fibers and foci.................................................................................................7 Large-scale screens reveal many additional foci and fibers .........................18 What’s going on? Three potential roles for intracellular bodies ...................21 Catalytic efficiency and improved regulation ......................................22 Storage depots ......................................................................................24 Aggregation of dysfunctional and/or unfolded proteins ......................25 Foundational Experiments ............................................................................26 S. cerevisiae GFP-strain library ...........................................................26 Cell-chip screen of stationary phase yeast GFP-library strains ...........27 Foundational experiments ....................................................................27 Future Chapters ....................................................................................30 Chapter 2: Widespread protein aggregation redefines cytoplasmic architecture in stress and aging .............................................................................................52 Introduction ...................................................................................................52 Results and Discussion .................................................................................55 Testing for multienzyme assemblies of pathways ...............................55 Heat shock causes similar sets of cytoplasmic proteins to become insoluble and form foci ...............................................................58 Co-immunoprecipitation of foci forming proteins ...............................61 Arsenic induced insolubility recapitulates stationary phase induced insolubility ..................................................................................65 Gln1-GFP foci formation correlates with aging ..................................66 vii Rapamycin treatment suppresses Gln1-GFP foci formation ...............69 Conclusion ....................................................................................................74 Methods.........................................................................................................75 References .....................................................................................................82 Supporting Information ...............................................................................102 Chapter 3: Protein aggregation as a widespread and conserved part of aging .....107 Introduction .................................................................................................107 Results and Discussion ...............................................................................109 Measuring protein insolubility in yeast during aging. .......................109 Conservation of age dependent protein insolubility between yeast and worm .........................................................................................114 A genetic link between thermotolerance and aging supports general aggregation as a common mechanism of senescence ...............116 Conclusion ..................................................................................................118 Methods.......................................................................................................118 References ...................................................................................................123 Supplemental Information ..........................................................................143 Chapter 4: Cellular Energy Buffers Protein Aggregation Propensity: Rappelling over the “edge”....................................................................................................145 Introduction .................................................................................................145 Results and Discussion ...............................................................................149 Widespread foci formation in S. pombe YFP screen .........................149 The sets of foci and fiber forming proteins in S. pombe and S. cerevisiae possess properties of aggregates ...............................................150 The S. cerevisiae glutamine synthetase (Gln1-GFP) as a model aggregate ...................................................................................................153 Formation of Gln1-GFP foci depends strongly upon cellular energy levels ...................................................................................................154 Gln1-GFP aggregates coalescence into single foci in an energy dependent manner.......................................................................................156 Conclusion ..................................................................................................161 Methods.......................................................................................................161 viii References ...................................................................................................164 Chapter 5: Discussion ..........................................................................................185
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