Understanding Mechanisms of Zinc Homeostasis in Schizosaccharomyces Pombe
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Understanding mechanisms of zinc homeostasis in Schizosaccharomyces pombe DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Sang‐Yong Choi The Ohio State University Nutrition Graduate Program The Ohio State University 2015 Dissertation Committee: Amanda J. Bird, Ph.D. (Advisor) Earl H. Harrison, Ph.D. Daren L. Knoell, Pharm.D. Kichoon Lee, Ph.D. Copyrighted by Sang‐Yong Choi 2015 Abstract Zinc is essential for cell growth, but can be toxic when in excess. As a consequence, intracellular zinc levels are tightly controlled by complex mechanisms that maintain zinc homeostasis in all biological creatures. In eukaryotic cells, factors that affect zinc homeostasis include zinc transporters, zinc buffering molecules, and zinc‐ regulatory factors. The levels of these factors are precisely regulated to maintain optimal intracellular zinc levels. To extend the growing literature in the roles of genes involved in zinc homeostasis, this dissertation investigates the role of the above executants in maintaining cytosolic zinc levels in Schizosaccharomyces pombe. In S. pombe, Loz1 plays a specific role in repressing gene expression when zinc is in excess. In zinc‐replete conditions, Loz1 down‐regulates zrt1, a gene encoding a high affinity zinc uptake transporter, and indirectly up‐regulates zym1 expression, a gene which encodes a zinc metallothionein. Deletion of Loz1, thus, causes constitutive zrt1 expression and low zym1 expression. In addition, cells lacking a functional loz1 gene hyperaccumulate zinc in zinc‐rich medium. In chapters 2 and 3, I take advantage of the loz1 mutation, and other yeast mutants to determine the roles of specific zinc transporters and zinc buffering molecules in maintaining zinc levels in the cytosol. ii Specifically, I utilize genetically encoded zinc‐responsive FRET‐based sensors, which allow changes in the labile pool of cytosolic labile zinc to be measured. My results show that Zhf1, a zinc transporter in endoplasmic reticulum membrane, and Zrg17 and Cis4, which reside in Golgi membrane, play a role in maintaining the cytosolic labile zinc pools upon a zinc shock. In addition, my works describe how Loz1 controls zinc homeostasis in zinc‐replete conditions and reveal that phytochelatins, small molecules that have a well‐ known role in the detoxification of toxic heavy metals, also have an important zinc buffering role. In chapters 4 and 5, I investigate the molecular mechanisms by which zinc‐ responsive transcription factors are regulated by zinc. Using chimeric proteins containing Loz1, this study examines the accessory domain adjacent to a double zinc finger, and shows that it is necessary for Loz1‐mediated zinc‐responsive changes in gene expression. This dissertation provides a platform for the understanding of zinc homeostasis mechanisms in fission yeast by examining the role that specific zinc transporters, zinc buffering molecules, and the zinc‐regulated factor Loz1 play in regulating cellular zinc levels. The results suggest that specific zinc transporters and Loz1 control the labile zinc pool in cells. Also, the roles of phytochelatins are highlighted as zinc buffering molecules. This discovery extends the current knowledge of how zinc buffering molecules influence metal homeostasis. While a number of zinc transporters have been identified in various iii eukaryotic cells, the zinc buffering molecules that modify the labile zinc pool remain to be further investigated. iv Acknowledgments My sincere gratitude goes to my advisor and mentor, Dr. Amanda Bird, for all her guidance and support throughout my doctoral studies. I would not have been able to complete all this without her kind assistance and optimistic view. I am thankful to all the members in Dr. Bird’s lab for their help as well. My dissertation committee members, Dr. Kichoon Lee, Dr. Daren Knoell, and Dr. Earl Harrison, have been a great support for my research. Also, my studies would not have been possible without the ZapCY1 and ZapCY2 constructs kindly provided by Dr. Amy Palmer at University of Colorado. I would like to thank Dr. Hyeyoung Kim at Yonsei University, who has been a warm and supportive teacher throughout the years. Finally, I would like to thank my mother, Seon‐Sook Yi, and father, Joong‐Suk Choi, for all their love and support from home. Also, I am grateful to Janice Jung for countless things. v Vita 2006 ...............................................................B.S. Food & Nutrition and Biology, Yonsei University, South Korea 2008 ...............................................................M.S. Food & Nutrition, Yonsei University, South Korea 2009 to present .............................................The Ohio State University Nutrition Program, The Ohio State University, USA Publications KM Ehrensberger, ME Corkins, S Choi, and AJ Bird. The double zinc finger domain and adjacent accessory domain from the transcription factor Loss of zinc sensing 1 (Loz1) are necessary for DNA binding and zinc sensing. J Biol Chem 2014 (289) 18087‐18096. S Choi and AJ Bird. Zinc'ing sensibly: controlling zinc homeostasis at the transcriptional vi level. Metallomics 2014 (6) 1198‐1215. YC Chan, J Banerjee, SY Choi, and CK Sen. miR‐210: the master hypoxamir. Microcirculation 2012 (3) 215‐223. SY Choi, JW Lim, T Shimizu, K Kuwano, JM Kim, and H Kim. Reactive oxygen species mediate Jak2/Stat3 activation and IL‐8 expression in pulmonary epithelial cells stimulated with lipid‐associated membrane proteins from Mycoplasma pneumoniae. Inflamm Res. 2012 (5) 493‐501. SY Choi, JH Yu, and H Kim. Mechanism of α‐lipoic acid‐induced apoptosis of lung cancer cells: Involvement of Ca2+. Ann NY Acad Sci. 2009 (1171) 149‐155. Fields of Study Major Field: The Ohio State University Nutrition Graduate Program vii Table of Contents Abstract ................................................................................................................................ii Acknowledgments ................................................................................................................ v Vita ..................................................................................................................................... vii Table of Contents ............................................................................................................. viiii List of Tables ..................................................................................................................... xiv List of Figures ..................................................................................................................... xv CHAPTER 1. Zinc in life ........................................................................................................ 1 1.1 Introduction ............................................................................................................ 1 1.2. Zinc in public health ............................................................................................... 2 1.2.1. Occurrence of zinc deficiency ...................................................................... 2 1.2.2. Nutritional intervention of zinc deficiency .................................................. 3 1.3. Biochemical functions of zinc ................................................................................ 5 1.3.1. Functions of zinc in metalloproteins ........................................................... 6 1.3.2. Zinc signaling ............................................................................................... 7 viii 1.4. Zinc homeostasis in mammals ............................................................................... 7 1.4.1. Zinc transporters ......................................................................................... 8 1.4.2. Metallothioneins (MTs) ............................................................................. 14 1.4.3. MTF‐1 ......................................................................................................... 15 1.5. Zinc homeostasis in S. cerevisiae ......................................................................... 20 1.5.1. Zinc transporters ....................................................................................... 21 1.5.2. Zap1 ........................................................................................................... 25 1.6. Zinc homeostasis in Schizosaccharomyces pombe .............................................. 28 1.6.1. Zinc transporters ....................................................................................... 29 1.6.2. Metal storage proteins/peptides .............................................................. 32 1.6.3. Loz1 ............................................................................................................ 34 1.7. Overview .............................................................................................................. 38 CHAPTER 2. Monitoring intracellular zinc distribution using FRET‐based genetic zinc sensors .............................................................................................................................. 40 2.1. Introduction ......................................................................................................... 40 2.2. Materials and methods ........................................................................................ 43 2.2.1. Yeast strains and growth conditions ........................................................