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The Pennsylvania State University The Graduate School Eberly College of Science REGULATION OF MITOCHONDRIAL TRANSLATION AND OXIDATIVE PHOSPHORYLATION THROUGH REVERSIBLE ACETYLATION A Dissertation in Biochemistry, Microbiology and Molecular Biology by Hüseyin Çimen 2012 Hüseyin Çimen Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2012 The Dissertation of Hüseyin Çimen was reviewed and approved* by the following: Emine C. Koc Assistant Professor of Biochemistry and Molecular Biology Dissertation Co-adviser Co-chair of Committee Hasan Koc Assistant Professor of Natural Sciences Dissertation Co-adviser Co-chair of Committee Craig E. Cameron Paul Berg Professor of Biochemistry and Molecular Biology Associate Department Head for Research and Graduate Education Joseph C. Reese Professor of Biochemistry and Molecular Biology Teh-hui Kao Professor of Biochemistry and Molecular Biology Tae-Hee Lee Assistant Professor of Chemistry and the Huck Institute of the Life Sciences Craig E. Cameron Paul Berg Professor of Biochemistry and Molecular Biology Associate Department Head of the Department of Biochemistry and Molecular Biology iii ABSTRACT In a eukaryotic cell, mitochondria provide energy in the form of ATP through oxidative phosphorylation (OXPHOS), which consists of five electron transport chain complexes embedded in the inner membrane of mitochondria. Human mitochondria have their own genome and transcription/translation system to synthesize mitochondrially encoded thirteen proteins of respiratory chain complexes. We investigated how acetylation of ribosomal proteins regulates translation and energy production in mitochondria since reversible acetylation of mitochondrial proteins was found to be critical for maintaining energy homeostasis. We identified mitochondrial ribosomal protein L10 (MRPL10) as the major acetylated ribosomal protein in mammalian mitochondria with two-dimensional gel electrophoresis followed by tandem mass spectrometry and immunoblotting analyses. In addition, we discovered that SIRT3, which is the main NAD+-dependent deacetylase localized into mitochondria, interacts with the ribosome and is responsible for the deacetylation of MRPL10. MRPL10 is a member of L7/L12 stalk, which is essential for translation since this stalk region stimulates the activity of translation factors. We employed SIRT3 knock-out mice in order to study the mechanism of reversible acetylation of MRPL10. The acetylation of MRPL10 resulted in increased MRPL12 binding to the L7/L12 stalk accompanied by enhanced protein synthesis in our in vitro translation assays. Moreover, HIB1B, a brown adipocyte tissue cell line stably overexpressing SIRT3, demonstrated reduction in the acetylation of MRPL10 and decreased MRPL12 binding to the L7/L12 stalk. By using [35S]-methionine pulse-labeling assays, we revealed that the mitochondrial protein synthesis was iv suppressed in these cells overexpressing SIRT3. Diminished synthesis of mitochondrial- encoded protein subunits of respiratory chain complexes resulted in reduced activities of Complex I and IV and total ATP production. Overall, these findings define a possible mechanism by which SIRT3-dependent reversible acetylation of MRPL10 and binding of MRPL12 to the ribosome regulates the mitochondrial protein synthesis and, therefore, modulates the OXPHOS and ATP production. In the next chapter, the discovery of another novel SIRT3 substrate, the flavoprotein (SdhA) subunit of Complex II, succinate dehydrogenase, was demonstrated. We identified and assessed the acetylation of the SdhA subunit, which resulted in reduced activity of Complex II in SIRT3 knock-out mice. Due to their location in the enzyme, the acetylated lysine residues may induce conformational changes to the active site of the enzyme in order to regulate its activity. Next, the advancement in the available expressed sequence tag (ESTs) databases from different organisms and the improved sensitivity of mass spectrometry-based proteomic studies encouraged us to reevaluate the protein components of the mammalian mitochondrial ribosome. In our analyses, we identified three additional members of the mitochondrial ribosome; CHCHD1, AURKAIP1, and CRIF1. We found that siRNA mediated knockdown of the newly identified ribosomal proteins to impair mitochondrial protein synthesis as determined by [35S]-methionine pulse-labeling assays. Overall, these biochemical and proteomic studies identified novel acetylated targets, MRPL10 and SdhA, for SIRT3. Given that the components of mitochondrial translation are crucial in the synthesis of respiratory chain subunits, the newly identified ribosomal proteins in addition to acetylation of MRPL10 and SdhA provide a more v complete picture of mitochondrial translation and regulation of energy production in the cell. vi TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................... ix LIST OF TABLES ....................................................................................................... xii ABBREVIATIONS ..................................................................................................... xiii ACKNOWLEDGMENTS ........................................................................................... xv Chapter 1 Introduction ................................................................................................ 1 1.1 Mitochondrion ................................................................................................ 1 1.2 Mitochondrial Genome ................................................................................... 4 1.3 Oxidative Phosphorylation ............................................................................. 7 1.4 Mitochondrial Translation .............................................................................. 8 1.4.1 Initiation ............................................................................................... 8 1.4.2 Elongation ............................................................................................. 9 1.4.3 Termination .......................................................................................... 10 1.5 Mitochondrial Ribosome ................................................................................ 12 1.5.1 Small Subunit (28S) of Mammalian Mitochondrial Ribosome ............ 15 1.5.2 Large Subunit (39S) of Mammalian Mitochondrial Ribosome ............ 15 1.5.3 New Mitochondrial Ribosomal Proteins and Additional Functions ..... 17 1.6 Post-Translational Modifications of Proteins ................................................. 20 1.6.1 Lysine Acetyltransferases (KATs) and Deacetylases (KDACs) .......... 20 1.6.2 Mitochondrial Acetyltransferases and Sirtuins .................................... 23 1.6.3 Post-Translational Modifications of Mitochondrial Ribosomal Proteins ................................................................................................... 28 1.7 Research Aims ................................................................................................ 29 1.8 References ....................................................................................................... 32 Chapter 2 Regulation of Mitochondrial Translation by SIRT3 .................................. 39 2.1 Rationale ......................................................................................................... 40 2.2 Introduction ..................................................................................................... 41 2.3 Materials and Methods ................................................................................... 45 2.3.1 Mitochondrial Ribosome Preparation and Reverse Phase – High Performance Liquid Chromatography (RP-HPLC). ............................... 45 2.3.2 Mass Spectrometric Analysis of Bovine Mitochondrial Ribosomal Proteins ................................................................................................... 46 2.3.3 In Vitro Deacetylation and Translation Assays .................................... 47 2.3.4 Mouse Mitochondrial Ribosome Isolation ........................................... 48 2.3.5 Immunoblotting Assays ........................................................................ 49 2.3.6 Plasmid Constructs ............................................................................... 50 2.3.7 Cell Culture .......................................................................................... 51 vii 2.3.8 [35S]-Methionine Pulse-Labeling Assays ............................................. 52 2.3.9 Preparation of Mitochondrial Ribosomes from Cell Lines .................. 52 2.3.10 Complex I and IV Activity and ATP Determination Assays ............. 53 2.3.11 Expression and Purification MRPL10-MRPL12 Complex and Reconstitution of Hybrid Ribosome ....................................................... 55 2.3.12 Statistical Analysis ............................................................................. 56 2.4 Results and Discussion .................................................................................. 57 2.4.1 The Mitochondrial Ribosomal Protein MRPL10 is Acetylated and SIRT3 is Responsible for its Deacetylation ........................................... 57 2.4.2 SIRT3, NAD+-dependent Deacetylase is Associated with 55S Mitochondrial Ribosome. ....................................................................... 66 2.4.3 Recombinant and Ribosome-Associated
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