Duquesne University Duquesne Scholarship Collection Electronic Theses and Dissertations Summer 2014 The Role of the B-Type Phospholipases in S. cerevisiae: Function, Regulation, and Physiological Relevance in Lipid Homeostasis Beth A Surlow Follow this and additional works at: https://dsc.duq.edu/etd Recommended Citation Surlow, B. (2014). The Role of the B-Type Phospholipases in S. cerevisiae: Function, Regulation, and Physiological Relevance in Lipid Homeostasis (Doctoral dissertation, Duquesne University). Retrieved from https://dsc.duq.edu/etd/1255 This Immediate Access is brought to you for free and open access by Duquesne Scholarship Collection. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Duquesne Scholarship Collection. For more information, please contact [email protected]. THE ROLE OF B-TYPE PHOSPHOLIPASES IN S. CEREVISIAE: FUNCTION, REGULATION, AND PHYSIOLOGICAL RELEVANCE IN LIPID HOMEOSTASIS A Dissertation Submitted to the Bayer School of Natural and Environmental Sciences Duquesne University In partial fulfillment of the requirements for the degree of Doctor of Philosophy By Beth A. Surlow August 2014 Copyright by Beth A. Surlow 2014 THE ROLE OF B-TYPE PHOSPHOLIPASES IN S. CEREVISIAE: FUNCTION, REGULATION, AND PHYSIOLOGICAL RELEVANCE IN LIPID HOMEOSTASIS By Beth A. Surlow Approved June 5, 2014 ________________________________ ________________________________ Dr. Jana Patton-Vogt Dr. Philip Auron Associate Professor of Biological Professor of Biological Sciences Sciences (Committee Member) (Committee Chair) ________________________________ ________________________________ Dr. Joseph McCormick Dr. Jeffrey Brodsky Chair and Associate Professor of Professor of Biological Sciences Biological Sciences University of Pittsburgh (Committee Member) (Committee Member) ________________________________ Dr. Philip Reeder Dean, Bayer School of Natural and Environmental Science iii ABSTRACT THE ROLE OF B-TYPE PHOSPHOLIPASES IN S. CEREVISIAE: FUNCTION, REGULATION, AND PHYSIOLOGICAL RELEVANCE IN LIPID HOMEOSTASIS By Beth A. Surlow August 2014 Dissertation supervised by Dr. Jana Patton-Vogt. Membrane phospholipid synthesis and turnover is a continual process during normal cell growth. The turnover of the glycerophospholipids by B-type phospholipases (PLBs) in Saccharomyces cerevisiae results in the formation glycerophosphodiesters through a deacylation reaction. Here, I address several aspects of the glycerophospholipid deacylation, transport, and reutilization pathway in S. cerevisiae. First, I show the RAS GTPase-activating proteins, Ira1 and Ira2, are required for utilization of the glycerophosphodiester – glycerophosphoinositol (GroPIns) – as a phosphate source. Second, I demonstrate loss of the cell surface associated PLBs, Plb1-3, and/or utilization of GroPIns causes actin cytoskeleton defects and an increased cell size. For the third and major part of my dissertation, I identified a novel interaction between Ypk1 and Plb1. Ypk1, the yeast homolog of the human serum- and glucocorticoid-induced kinase (Sgk1), affects diverse cellular activities, including sphingolipid homeostasis. Here, I report that Ypk1 also iv impacts the turnover of the major phospholipid, phosphatidylcholine (PC). Pulse-chase radiolabeling reveals that a ypk1∆ mutant exhibits increased Plb1-mediated PC deacylation and glycerophosphocholine (GroPCho) production compared to wild type. Consistent with a link between Ypk1 and Plb1, the levels of both Plb1 protein and PLB1 message are elevated in a ypk1∆ strain compared to WT yeast. Furthermore, I discovered that an increase in PLB1 expression also occurs upon disruption to sphingolipid synthesis and is mediated by the Crz1 transcription factor. Taken together, these findings suggest that sphingolipid synthesis is coordinated with PC turnover to maintain optimal lipid homeostasis. v ACKNOWLEDGEMENT Thank you to my advisor Dr. Jana Patton-Vogt for giving me the opportunity to work on this project, and for her continual guidance, advice, and support of both my research, and teaching endeavors. She has guided my development as a scientist in basic lab skills, scientific communication, and in problem solving/reasoning. Thank you to my outside committee member, Dr. Jeff Brodsky, for his excellent support and advice, and for allowing me to learn a few techniques in collaboration with his lab. I also want to thank my committee members, Dr. Auron and Dr. McCormick for all of their helpful suggestions and for their time spent both during and outside my committee meetings assisting in various aspects of this project. I want to acknowledge all of the members of the Patton-Vogt laboratory, past and present, for helpful conversations, both scientific and personal, during the time we shared together in the lab, including Andrew Bishop, Kellie Rosiek, Tao Sun, Nicole Nesbitt, Sanket Anaokar, and all of the undergraduates I have worked with, especially thank you to Ben Cooley and Carole Wolfe who have processed many samples and worked independently in the lab. I am especially grateful for Andy, who helped train me initially when I joined the lab, and who went through most of the grad school experience with me. I truly appreciate the working relationship and friendship. I am grateful for all the friendships and colleagues I have made with my fellow graduate students. Thank you especially to Tiffaney Czapski, as we worked through many of the rough patches together. I appreciate all of the scientific and social adventures with all of you, Andy, Tiff, Kellie, Sarah, Metis, Natasha, Maria, Sumedha, Chris, Allen, Stephanie, Andre, Heather, Nick, AnJey, Juraj, and Ruth. A special thank you to all of my family who encouraged me in continuing this journey without giving up, especially my parents, in-laws, and siblings – Mom, Dad, Caren, John, Marie, vi Vince, Sarah, and Abby. I truly appreciate all of your help especially the last two years since Heidi was born. You have each graciously volunteered to help with babysitting, laundry, cooking, etc. exactly when the extra pair of hands was most needed, and have of course provided much emotional support. A special thank you also to my grandma and late grandpa who have said countless prayers for me. I am very fortunate and thankful to have a wonderful, supportive family! Most importantly, I want to thank my husband, Will. Your daily support over the last six plus years, and your patience, understanding, and love through all the ups and downs is greatly appreciated. Your encouragement to keep going was my motivation. Thank you for being both the dad and the “mom” for Heidi at times, especially during the past few months. And lastly, thank you to my daughter, Heidi. I can always depend on you to turn my frown upside down. Mommy is “all done” with school and I will be spending more time with you and daddy soon! vii TABLE OF CONTENTS Page Abstract .......................................................................................................................................... iv Acknowledgement ......................................................................................................................... vi List of Tables ................................................................................................................................ xii List of Figures .............................................................................................................................. xiii List of Abbreviations .....................................................................................................................xv Chapter 1 Introduction .....................................................................................................................1 1.1 Saccharomyces cerevisiae as a model organism ................................................................................. 1 1.2 Major lipid classes in the S. cerevisiae plasma membrane ................................................................. 1 1.3 Glycerophospholipid metabolism in yeast .......................................................................................... 4 1.4 Role and classification of phospholipases........................................................................................... 9 1.5 Glycerophosphodiester production and transport in S. cerevisiae and higher eukaryotes ................ 12 1.6 The B-type Phospholipases in S. cerevisiae ...................................................................................... 15 1.7 Glycerophosphodiesterase hydrolysis of the glycerophosphodiesters .............................................. 17 1.8 Summary of Background and Significance....................................................................................... 17 Chapter 2 Neurofibromin Homologs Ira1 and Ira2 affect glycerophosphoinositol Production and Transport in Saccharomyces cerevisiae .........................................................................................19 2.1 Introduction ....................................................................................................................................... 20 2.2 Results and Discussion ..................................................................................................................... 21 2.2.1 Identification of IRA genes as affecting GroPIns metabolism. .................................................. 21 2.2.2 An ira1∆ira2∆ mutant exhibits altered GroPIns transport activity. ..........................................