University of South Carolina Scholar Commons Theses and Dissertations 2015 Illuminating the Interactions and Functions of Glutaredoxins, BolA Proteins, and Erv1 in Iron Homeostasis Adrian Colleen Dlouhy University of South Carolina - Columbia Follow this and additional works at: https://scholarcommons.sc.edu/etd Part of the Chemistry Commons Recommended Citation Dlouhy, A. C.(2015). Illuminating the Interactions and Functions of Glutaredoxins, BolA Proteins, and Erv1 in Iron Homeostasis. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/3193 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]. ILLUMINATING THE INTERACTIONS AND FUNCTIONS OF GLUTAREDOXINS, BOLA PROTEINS, AND ERV1 IN IRON HOMEOSTASIS by Adrienne Colleen Dlouhy Bachelor of Science North Carolina State University, 2009 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 Outten, Major Professor John Dawson, Committee Member Andrew Greytak, Committee Member Erin Connolly, Committee Member Lacy Ford, Vice Provost and Dean of Graduate Studies © Copyright by Adrienne Colleen Dlouhy, 2015 All Rights Reserved. ii ACKNOWLEDGEMENTS First, I would like to acknowledge my advisor, Dr. Caryn Outten. She has been helpful and encouraging throughout my time in her lab, even when it seemed like none of my experiments were working. She’s given me many great opportunities to learn and grow as a scientist. I would also like to thank my committee members, Dr. John Dawson, Dr. Andrew Greytak, and Dr. Erin Connolly for all of their support and advice. Additionally, I would like to thank all of my Coutten (and Woutten) labmates, past and present: Max Darch, Khaleh Thomas, Sam Bouldin, Angela-Nadia Albeţel, John Hepburn, Haoran Li, Kirsten Collins, Hatice Ozer, Crystal Conway, Malini Gupta, Rabi Behera, Jingjing Hu, Nin Dingra, and Zuqin Xue. In particular, Henry for getting me started in the lab and setting the bar high. Sam for taking me under her wing and being there for me, even after moving to Tennessee. Angela for trying to teach me EPR and to be nicer. John for all the snacks and talks while I was running AA. And especially Max and Khaleh for keeping me sane and sticking it out together for six years. I would also like to thank all the other graduate students and postdocs who helped along the way when they could, listening to talks, reading abstracts, and showing me the ropes. Finally, I would like to thank my family and all of my non-graduate school friends for being encouraging, even when they had no idea what I was talking about. They are unwavering in their support and always have my back. And I love them even though they each asked “When are you graduating?” at least 16 times. iii ABSTRACT Iron is a redox-active protein cofactor required for essential cellular functions such as respiration, however excess intracellular iron can generate damaging reactive oxygen species. Understanding how cells regulate iron levels is critical for treatment of human diseases that span from anemia to iron overload disorders. Glutaredoxins (Grxs) with a CGFS active site are highly conserved proteins shown to have roles in iron homeostasis and iron-sulfur cluster assembly, thus earning them the title “the Iron Whores”. They can exist either as a [2Fe-2S] cluster-bound dimer or an apo monomer, suggesting conservation of structure and function. In addition, Grxs interact with the BolA family of proteins, which also have genetic connections to metal and sulfur metabolism. In the model eukaryote Saccharomyces cerevisiae, CGFS-type Grxs and the BolA-like protein Fra2 were shown to transfer an Fe-S cluster to the transcriptional activators Aft1 and Aft2, inhibiting their DNA binding activity. E. coli express one CGFS glutaredoxin, Grx4, and two BolA-like proteins, BolA and YrbA. Grx4 forms [2Fe-2S]-bridged homodimers alone, while co-expression of Grx4 with BolA or YrbA yields [2Fe-2S]-bridged heterodimers. In vitro studies indicate differences in Fe-S cluster binding between these two heterodimers. These results reinforce the idea that Grx4 acts as Fe-S transport and delivery proteins, while interaction with BolA/YrbA may alter the function or specificity. iv In the fission yeast Schizosaccharomyces pombe, the CGFS glutaredoxin Grx4 interacts with and regulates the iron-dependent transcriptional repressor Php4. Similar to its homologues, Grx4 forms a [2Fe-2S]-bridged homodimer alone, and a [2Fe-2S]-bridged heterocomplex when co-expressed with Php4. Comparison of these complexes indicates differing cluster coordination environments. These results suggest that when iron is sufficient, Grx4 interacts with Php4 to form a [2Fe-2S] cluster-bound complex, communicating cellular iron status and inhibiting Php4 activity. Erv1 is a sulfhydryl oxidase involved in importing proteins into the mitochondria. In S. cerevisiae, Erv1 was implicated in cytosolic Fe-S cluster proteins maturation and iron regulation. However, these studies were performed on a single erv1 mutant strain, erv1-1, that we discovered has additional defects in glutathione metabolism. To investigate the Erv1-dependent connection between GSH metabolism and iron homeostasis, we measured GSH levels and Fe-S protein activity in a variety of erv1 mutants. Only the erv1-1 strain has significantly reduced GSH levels, due to a mutation in a glutathione biosynthesis gene. This mutation causes dysfunctional iron regulation and iron accumulation. Together, these results suggest that the Fe-S cluster maturation and iron regulation defects reported in the erv1-1 strain are due to a mutation in GSH metabolism, rather than indicating a direct role for Erv1 in iron metabolism. v TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................................ iii ABSTRACT .......................................................................................................................... iv LIST OF TABLES .................................................................................................................. ix LIST OF FIGURES ...................................................................................................................x LIST OF ABBREVIATIONS ................................................................................................... xiii CHAPTER 1: INTRODUCTION AND SCOPE OF THESIS ..............................................................1 INTRODUCTION AND SIGNIFICANCE .............................................................................1 PROPERTIES OF THE IRON METALLOME .......................................................................2 IRON METALLOPROTEINS ..........................................................................................12 IRON UPTAKE, TRAFFICKING, AND STORAGE ............................................................29 REGULATION OF IRON ...............................................................................................42 CONCLUSIONS ...........................................................................................................58 SCOPE OF THESIS .......................................................................................................59 COPYRIGHT RELEASE ................................................................................................62 CHAPTER 2: BOLA AND YRBA FORM DISTINCT FE-S CLUSTER COMPLEXES WITH GRX4 IN E. COLI .................................................................................................................67 ABSTRACT .................................................................................................................67 INTRODUCTION ..........................................................................................................68 MATERIALS AND METHODS .......................................................................................71 RESULTS AND DISCUSSION ........................................................................................77 vi CONCLUSIONS .........................................................................................................110 CHAPTER 3: GRX4 REGULATES PHP4 FUNCTION VIA FE-S CLUSTER BINDING IN S. POMBE ................................................................................................................113 ABSTRACT ...............................................................................................................113 INTRODUCTION ........................................................................................................114 MATERIALS AND METHODS .....................................................................................118 RESULTS ..................................................................................................................122 DISCUSSION .............................................................................................................143 CHAPTER 4: IDENTIFYING FUNCTIONS OF BOLA-LIKE PROTEINS IN S. CEREVISIAE ...........147 ABSTRACT ...............................................................................................................147 INTRODUCTION ........................................................................................................148