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Functional Analysis of Cytosolic Hsp70 Nucleotide Exchange Factor Networks in Yeast The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 5-2014 Functional Analysis of Cytosolic Hsp70 Nucleotide Exchange Factor Networks in Yeast Jennifer Lynn Abrams Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Biochemistry Commons, Cell Biology Commons, Microbiology Commons, and the Molecular Biology Commons Recommended Citation Abrams, Jennifer Lynn, "Functional Analysis of Cytosolic Hsp70 Nucleotide Exchange Factor Networks in Yeast" (2014). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 447. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/447 This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. FUNCTIONAL ANALYSIS OF CYTOSOLIC HSP70 NUCLEOTIDE EXCHANGE FACTOR NETWORKS IN YEAST by Jennifer Lynn Abrams APPROVED: _____________________________________________ Supervisory Professor, Kevin A. Morano, Ph.D. _____________________________________________ Heidi B. Kaplan, Ph.D. _____________________________________________ Michael C. Lorenz, Ph.D. _____________________________________________ Eric J. Wagner, Ph.D. _____________________________________________ Ambro van Hoof, Ph.D. ___________________________________________________ Dean, The University of Texas Graduate School of Biomedical Sciences at Houston FUNCTIONAL ANALYSIS OF CYTOSOLIC HSP70 NUCLEOTIDE EXCHANGE FACTOR NETWORKS IN YEAST A DISSERTATION Presented to the Faculty of The University of Texas Health Science Center at Houston and The University of Texas MD Anderson Cancer Center Graduate School of Biomedical Sciences in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY by Jennifer Lynn Abrams, B.S. Houston, Texas May 2014 DEDICATION I would like to dedicate this thesis to my late Aunt Sally A. Malter. She was always supportive of me advancing my education and encouraged me to keep pushing forward when life was difficult. She will be greatly missed. iii ACKNOWLEDGMENTS The completion of my thesis has occurred only with the help and support of many people. First I would like to express my gratitude to my wonderful mentor, Dr. Kevin Morano, who has supported me and taught me so much over the years. His ability to keep me motivated, while simultaneously identifying ways I could improve as a scientist, is unmatched. He has made my time here enjoyable, but challenging, which has helped me to develop the skills necessary to move on to the next step in my career. I would also like to thank all of the members of the Morano lab past and present including Dr. Patrick Gibney, Dr. Hugo Tapia, Dr. Jacob Verghese, Dr. Yanyu Wang, Kim Cope, Veronica Garcia, and Sara Peffer. I could not have asked to work with a better group of people. All of the lab outings were a lot of fun in the company of these awesome people. In addition, engaging lab meetings and scientific discussions kept me on my toes. Not to mention all of the impromptu question/answer sessions prior to my candidacy exam that made facing my committee much easier. A special thanks to Sara Peffer, who I have know for the shortest time, but has always provided me with a smiling face and a clever gif just when I needed it. In addition, I would specifically like to thank Veronica Garcia, also known as “my other half”, for supporting me through good times and bad and for being an incredible friend inside and outside of the lab. I would also like to give a special thanks to Patrick Gibney, who mentored me through my rotation and has continued to be an extremely encouraging, wonderful person that I would not have made it this far without. I would like to acknowledge my committees members throughout the years that have provided me with the feedback and knowledge that was necessary for me to advance to this stage of my career. These members include: Dr. Heidi Kaplan, Dr. Michael Lorenz, Dr. Michael Blackburn, Dr. Ambro van Hoof, Dr. Hung Ton-That, and Dr. Eric Wagner. This engaging group of people guided me through my project and through graduate school regardless of their status on my committee. iv In my time here, I was lucky enough to receive awards that afforded me opportunities I may not have had otherwise. I would like to thank the American Society for Microbiology, who awarded me with the Robert. D. Watkins Graduate Research Fellowship. With this fellowship, I was able to attend the ASM General Meetings as well as the ASM minority conferences, which has helped build a network of people that I will keep in touch with throughout my career. In addition, I would like to thank the Graduate School for Biomedical Sciences (GSBS) for choosing me to receive the esteemed R.W. Butcher award that recognized my work for being a citizen’s scientist, which is an important aspect of who I am. My sincerest thanks goes to the Department of Microbiology and Molecular Genetics office staff, with special recognition for Lyz Morrison, Vicci Sanders, and Karen Oandasan, who all helped me work through various issues I faced throughout Graduate School. In addition, these ladies were always friendly and helpful even if I just needed to talk. I appreciate all the effort and care that went into maintaining a well-organized program and department. In addition, the faculty and staff at GSBS including: Joy Lademora, Bunny Perez, Dr. Andrew Bean, Brenda Gaughan, Eric Solberg, and Karen Weinberg have all helped me at different stages of my time here. The Microbiology and Molecular Genetics Program provided me with a very unique experience in that I have always felt so much support and love from everyone making it feel like a home away from home. I have made so many friends in this program from the very beginning. The ladies that I started classes with in 2007, Dr. Ale Klauer-King and Jennifer Herricks, were my study partners, my friends, and my counselors when I needed it, so I would like to thank them for that. In addition, Dr. Jenny Dale was a dedicated friend that helped me through every step of my candidacy exam. I have also met many lifelong friends here including Alex Marshall, Jill Losh, and Kim Busiek, who I have spent a lot of time getting to know and love. I met Katie “Squirt” McCallum in her rotation in my laboratory and she has become one of my best friends and is one of the most caring, supportive, and loving friends that I have. I would also like to thank the Valhalla crew including Katie McCallum, Veronica Garcia, George v Tiller, and Amy Ford; many difficult days were made better with the support of these people. Finally I would like to thank my tech support and good friend Doug Litwin for being there when I needed him and sometimes when I did not. I also have a number of great friends who have supported me outside of work that always listened to me no matter how much I wanted to talk about science; these people include Reisha Beaty, Joeseph Edmonson, both of whom I have known and have been incredibly supportive since high school as well as some new friends Matt Madeiro, Carissa Ara, Clint Stengel, Lauren Chapman, and Ambri Garcia who have all been accepting, loving friends. Finally, I would like to give a special thanks to Carlos Calderon, who has not only been supportive as a friend for many years, but has also provided a very conducive and encouraging environment for me to work and study, so this thesis could come to fruition. Aside from work I would like to thank my family who helped me overcome many obstacles and for whom none of this would be possible. I would first like to thank my parents Marion and James Abrams, who have always been my biggest supporters and have never doubted that I could do anything I wanted to with my life. I would also like to thank my brother Christopher Abrams, who always kept me grounded through the best times and the worst times. All of my family’s love and support has made me the person I am today and without them I could not have been successful in a career that I love. vi FUNCTIONAL ANALYSIS OF CYTOSOLIC HSP70 NUCLEOTIDE EXCHANGE FACTOR NETWORKS IN YEAST Jennifer Lynn Abrams, B.S. Advisory Professor: Kevin A. Morano, Ph. D. The Hsp70 class of molecular chaperones play critical roles in protein homeostasis via an ATP-dependent folding cycle. Cytosolic Hsp70s in the budding yeast Saccharomyces cerevisiae, Ssa and Ssb, interact with up to three distinct nucleotide exchange factors (NEFs) homologous to human counterparts; Sse1/Sse2/HSP110, Fes1/HspBP1, and Snl1/Bag1. In an effort to understand the differential functional contributions of the cytosolic NEFs to protein homeostasis (“proteostasis”), I carried out comparative genetic, biochemical and cell biological analyses. For these studies, I developed protocols to monitor protein disaggregation and reactivation in a near real-time coupled assay that revealed the importance of aggregate dynamics in the solubilization of proteins for their refolding. This coupled experimental approach, represents an important step toward developing tools necessary to monitor in vivo mechanisms of proteostasis. This work determined that the Hsp110, Sse1, is the primary NEF contributing to most Hsp70 functions and also uncovered a unique role for Fes1 in Ssa-mediated regulation of the cytosolic heat shock response, while revealing no significant contributions from Snl1 and Sse2.
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