University of South Carolina Scholar Commons Theses and Dissertations 2015 The Role of Intermembrane Space Redox Factors In Glutathione Metabolism And Intracellular Redox Equilibrium Hatice Kubra Ozer University of South carolina Follow this and additional works at: https://scholarcommons.sc.edu/etd Part of the Chemistry Commons Recommended Citation Ozer, H. K.(2015). The Role of Intermembrane Space Redox Factors In Glutathione Metabolism And Intracellular Redox Equilibrium. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/3702 This Open Access Dissertation is brought to you by Scholar Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. THE ROLE OF INTERMEMBRANE SPACE REDOX FACTORS IN GLUTATHIONE METABOLISM AND INTRACELLULAR REDOX EQUILIBRIUM by Hatice Kubra Ozer Bachelor of Science Uludag University, 2004 Master of Food Science and Nutrition Clemson University, 2010 Submitted in Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy in Chemistry College of Arts and Sciences University of South Carolina 2015 Accepted by: Caryn E. Outten, Major Professor F. Wayne Outten, Committee Chair Erin Connolly, Committee Member Andrew B. Greytak, Committee Member Lacy K. Ford, Senior Vice Provost and Dean of Graduate Studies © Copyright by Hatice Kubra Ozer, 2015 All Rights Reserved. ii ACKNOWLEDGEMENTS First, I would like to thank my advisor, Dr. Caryn E. Outten for her patience, guidance, expertise, and confidence in me to complete the work contained herein. She has been an excellent mentor during my graduate program. She is also the only person beside myself who is guaranteed to have read every word of this manuscript and to review every presentations in the past and her insight was irreplaceable. I would like to thank my committee members: Dr. F. Wayne Outten, Dr. Erin Connolly, and Dr. Qian Wang for their valuable input on my plan, and proposal, for helping me to fulfill the requirements for a Ph.D. in Chemistry. Also, a special thanks to Dr. Andrew Greytak for joining my dissertation defense committee at the last minute to replace Dr. Wang. Thanks to all the members of the Outten lab present and past (Dr. Vidyadhar Daithankar, Dr. Angela-Nadia Albetel, Crystal Conaway, Adrienne Dlouhy, Maxwell Darch, John S Hepburn, Malini Gupta, Kirsten R Collins, Naimah Bolaji, Matthew Blahut, Rabindra Behera, Yuyuan Dai, Suning Wang, Haoran (Henry) Li, and Zuqin Xie,) for their support, discussions, and advice. I owe to thanks to Henry and Rabi for all he taught me with huge patience and for helpful discussions. I would especially like to thank Vidy, Angela, Crystal, Naimah, Yuyuan, and Suning for their friendship and appreciate the contributions they have made toward my research. iii I would like to thank to my husband, Ufuk Ozer for his love, patience, great support, and for being there for me whenever I needed somebody to listen the complaints about my failures in research. My research would not have been possible without the support of him. I would like to thank to my parents, Mevlut and Hacer Tokpunar, have loved and stood with me my entire life, and for encouraging me to pursue a career in Biochemistry. Thanks to my mother in-law, Yeter Ozer for their love, support, taking good care of my children during my school years. Finally, I would like to acknowledge the Ministry of Education, Turkey for awarding me the Graduate Study Abroad Scholarship in USA. iv ABSTRACT The mitochondrial intermembrane space (IMS) is a unique subcellular compartment that houses key thiol-dependent redox pathways such as protein transport, mitochondrial respiration, and detoxification of ROS (reactive oxygen species). These pathways are all dependent on cysteine-rich proteins, thus maintaining thiol-disulfide balance in this organelle is crucial for cellular functions. An IMS protein import pathway called the Mia40-Erv1 disulfide relay system uses disulfide bond formation for the import and retention of substrate proteins in the IMS. Erv1 is also suggested to be involved in maturation of cytosolic Fe-S cluster proteins and regulation of iron homeostasis in S. cerevisiae. However, these studies were performed on one particular erv1 mutant strain (named as erv1-1) that we discovered has additional defects in glutathione (GSH) metabolism due to a secondary mutation in the gene encoding the GSH biosynthesis enzyme, Gsh1. Since the tripeptide GSH is also required for iron homeostasis and cytosolic Fe-S protein biogenesis, the Erv1-dependent connection between mitochondrial protein import, GSH metabolism, and iron homeostasis was investigated in several erv1 mutants. The GSH depletion phenotype was only detected in the erv1-1 strain and could be rescued by expressing GSH1 or adding GSH to the growth media. Additionally, expression of the iron uptake gene, FET3 and enzyme activities of Fe-S cluster proteins in several erv1 mutants were tested. Only the erv1-1 mutant has an iron misregulation defect, which could be rescued with GSH addition, and no significant effects on Fe-S cluster protein activities were detected. Our data suggests that the defects of cytosolic Fe- v S maturation and iron regulation first reported in the erv1-1 strain is a direct consequence of GSH depletion rather than indicating a direct role for Erv1 in iron metabolism and cytosolic Fe-S cluster biogenesis. We also characterized how mutations of Mia40 influence GSH metabolism and GSH:GSSG pools in the cytosol, mitochondrial matrix and intermembrane space. We have found that defects in Mia40 only influence the IMS redox state and do not alter cellular GSH levels. Additionally, we determined that defects in Mia40 do not impact iron homeostasis or Fe-S cluster biogenesis. Furthermore, utilizing the roGFP2 in vivo sensors, we demonstrated how the deletion of manganese cofactor of superoxide dismutase 2 transporter Mtm1 and the citrate-oxoglutarate carrier Yhm2 affect the redox status of the mitochondrial matrix and IMS and cellular GSH levels. Deletion of MTM1 only leads to a large oxidative shift in the IMS GSH:GSSG redox state. In the contrary, deletion of YHM2 shows a smaller effect on the mitochondrial GSH:GSSG redox state even though, both mtm1∆ and yhm2∆ mutants were shown to have reduced mitochondrial GSH levels. Overall, we successfully characterized the roles of Erv1 and Mia40 in GSH metabolism, mitochondrial import and subcellular redox state which hereby helps to reveal their roles in Fe-S cluster biogenesis and iron regulation. vi TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................................ iii ABSTRACT ........................................................................................................................... V LIST OF TABLES ................................................................................................................... iX LIST OF FIGURES .................................................................................................................. X LIST OF ABBREVIATIONS ................................................................................................... Xiii CHAPTER 1. INTRODUCTION .................................................................................................1 CELLULAR DISTRIBUTION OF THIOL REDOX SYSTEMS IN SACCHAROMYCES CEREVISIAE ................................................................................................................2 IN VIVO GENETICALLY ENCODED REDOX SENSORS .....................................................8 GSH , ERV 1-MIA 40 IMS DISULFIDE RELAY SYSTEM AND OTHER REDOX CONTROL SYSTEMS IN THE MITOCHONDRIAL IMS ......................................................................9 FE-S BIOGENESIS , IRON HOMEOSTASIS AND THEIR LINK WITH ERV 1 AND GSH .........15 SCOPE OF THESIS .....................................................................................................20 CHAPTER 2. THE ROLE OF ERV 1 IN GLUTATHIONE METABOLISM , CYTOSOLIC FE-S CLUSTER MATURATION AND IRON REGULATION ................................................................24 ABSTRACT ..............................................................................................................25 INTRODUCTION .......................................................................................................26 EXPERIMENTAL PROCEDURES .................................................................................29 RESULTS .................................................................................................................37 DISCUSSION ............................................................................................................60 CHAPTER 3. MUTATIONS IN MIA 40 AFFECTS THE IMS REDOX STATE AND HAVE NO EFFECTS ON IRON REGULATION ..........................................................................................65 ABSTRACT ..............................................................................................................66 INTRODUCTION .......................................................................................................67 EXPERIMENTAL PROCEDURES .................................................................................71 vii RESULTS .................................................................................................................74 DISCUSSION ............................................................................................................83