Identification of a novel biogenesis factor for mitochondrial Complex I using Chlamydomonas reinhardtii as a model system DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Nitya Subrahmanian Graduate Program in Plant Cellular and Molecular Biology The Ohio State University 2015 Dissertation Committee: Patrice P. Hamel, Advisor Anita K. Hopper Venkat Gopalan R. Keith Slotkin Copyright by Nitya Subrahmanian 2015 ABSTRACT Mitochondria, the “powerhouse” of the cell, produce energy through the mitochondrial respiratory chain, which comprises five major complexes. Complex I is the first and most complicated enzyme required in this process. With more than 40 subunits, one FMN molecule and eight Fe-S clusters, the assembly of mitochondrial Complex I is highly intricate. Complex I deficiency in humans causes severe metabolic defects leading to fatal diseases such as encephalomyopathy and Parkinson’s disease. In 60% of the patients presenting Complex I dysfunction, there is no molecular explanation and it is assumed that defects in yet-to-be discovered assembly factors are responsible for the Complex I deficiency. In this study, we present the identification of a novel factor required for the biogenesis of Complex I in a green alga. Study of Complex I biogenesis in humans is very limited due to the lethality of patients with Complex I-related diseases and associated ethical concerns. Chlamydomonas reinhardtii, a unicellular photosynthetic alga, is an ideal model organism for the study of Complex I assembly and function. Chlamydomonas is genetically tractable and it’s Complex I subunit composition is similar to its human counterpart. Unlike mammalian systems, Complex I deficient mutants in ii Chlamydomonas are viable due to their photosynthetic ability. Importantly, Chlamydomonas is the only model system whose nuclear and mitochondrial genomes can be easily manipulated for the study of mitochondrial Complex I. The major goal of the study presented in this thesis is to understand the biogenesis of mitochondrial Complex I using Chlamydomonas reinhardtii as a model. In Chapter 2, we describe a forward genetic screen, performed in Chlamydomonas reinhardtii, to identify novel loci controlling Complex I biogenesis. Six Complex I mutants amc8-to-amc13 (assembly of mitochondrial complex I) were isolated from this screen. In Chapter 3, we determine that the AMC9 locus is defined by the gene encoding a core 24 kDa Complex I subunit, NUO5. Conserved from bacteria to humans, this subunit is essential for the NADH oxidation activity of Complex I. In Chapter 4, we report the identification of a novel factor, AMC11, involved in Complex I biogenesis. We show that AMC11 is targeted to the mitochondria through its N-terminal sequence and that loss of AMC11 affects mitochondrial gene expression, resulting in a Complex I assembly defect. Finally, in Chapter 5, we describe the molecular characterization of additional amc mutants that display reduced levels of assembled Complex I. We describe that, although they maintain the structural integrity of Complex I, these mutants are associated with changes in mitochondrial transcript abundance. Overall, our study is the first to identify a novel Complex I biogenesis factor using a forward genetic screen. iii Dedication To my beloved family iv ACKNOWLEDGMENTS I sincerely thank my PhD Advisor Dr. Patrice Hamel for his constant guidance and support. He has truly been a very patient mentor and a source of constant encouragement through obstacles in research. Thank you for always reminding me that science is fun! I am also grateful to my committee members Dr. Richard Keith Slotkin, Dr. Venkat Gopalan and Dr. Anita Hopper for their enthusiasm and guidance in my research. I also thank Dr. Birgit Alber for extending her support by unfailingly attending all my research presentations and for providing advice. I extend my gratitude to the Hamel laboratory, especially Drs. Rosario Barbieri and Sara Cline for helping me through the initial years of graduate school. I also thank all the students, who worked with me in the Hamel laboratory, for helping me evolve into a better teacher. I am grateful to Dr. Claire Remacle, University of Liège, Belgium, for being a wonderful host and mentor during my visit to her laboratory. … I would also like to take this opportunity to express my heartfelt gratitude to my parents, Mr. B. S. Subrahmanian and Mrs. Parvathy Subrahmanian, whose love, encouragement and guidance have been very crucial for all my successes. I also extend this gratitude to my brother, Raju, and sister-in-law Rohini for their v constant support, advice and relaxing weekend getaways. I am also grateful to my baby nephew Srihari, for delighting me with his giggles. Last, but not the least, I thank all my friends in Columbus for making this city my home away from home. I would also like to thank Dr. Aishwarya Jacob and Megha Chandrashekhar, you were always my friends in need! vi Vita May 2004 ................................................ P.S.B.B.S.S.School, India. June 2008 ............................................... B.Tech Biotechnology, VIT University. 2008 to present ...................................... Graduate Teaching and Research Associate, Department of Plant Cell and Molecular Biology, The Ohio State University. PUBLICATIONS 1. Remacle C, Larosa V, Salinas T, Hamel P, Subrahmanian N, Bonnefoy N, Kempken F: Transformation and nucleic acid delivery to mitochondria. In: Bock C, Knoop V (eds) Genomics of chloroplasts and mitochondria, Advances in photosynthesis and respiration, (2012), vol 35, ISBN 978-94- 007-2919-3. 2. Remacle C, Hamel P, Larosa V, Subrahmanian N, Cardol P Complexes I in the green lineage. In: Sazanov L (ed) A structural perspective on vii respiratory complex I, (2012) ISBN 13: 9789400741379, ISBN 10: 9400741375 3. Barbieri MR, Larosa V, Nouet C, Subrahmanian N, Remacle C, Hamel PP: A forward genetic screen identifies mutants deficient for mitochondrial complex I assembly in Chlamydomonas reinhardtii. Genetics 188: 349-58 (2011). FIELDS OF STUDY Major Field: Plant Cellular and Molecular Biology viii Table of Contents Abstract………………………………………………………. ................................... ii Acknowledgments………………………………………….. ................................... v Publications…………………………………………. ............................................ vii Fields of Study……………………………………….. ......................................... viii LIST OF Tables………………………………………. ........................................... xv LIST OF Figures………………………………………………….. ........................ xvii CHAPTER 1………………………………………………. ........................................ 1 INTRODUCTION………………………………….. .................................................. 1 1.1. Mitochondrial Electron Transfer Chain. ................................. 1 1.2. Mitochondrial Complex I .......................................................... 2 1.3. Core subunits ........................................................................... 4 1.4. Non-core subunits of Complex I ................................................ 10 1.4.1. GRIM-19 (Gene associated with Retinoic IFN induced Mortality-19) . 11 1.4.2. Acyl carrier protein (SDAP/ NDUFAB1) ............................................. 12 1.4.3. γ - Carbonic Anhydrase (CA/CAL) subunits ....................................... 13 1.4.4. GLDH (L-galactano, 1,4-lactone dehydrogenase) .............................. 15 1.4.5. Post-translational modification of Complex I subunits ........................ 16 1.5. The assembly of Complex I ................................................... 18 ix 1.6. Assembly Factors for Mitochondrial Complex I .................. 20 1.6.1. C20ORF7 / NDUFAF5 ...................................................................... 22 1.6.2. C6ORF60 / NDUFAF3 and C6ORF66 / NDUFAF4. .......................... 22 1.6.3. C8ORF38 / NDUFAF6 ...................................................................... 22 1.6.4. CIA30 / NDUFAF1 ............................................................................. 23 1.6.5. Ecsit (Evolutionarily conserved signal intermediate in Toll pathways)……. .................................................................................. 24 1.6.6. ACAD9 (Acyl CoA dehydrogenase) .................................................. 24 1.6.7. TMEM126B ....................................................................................... 25 1.6.8. C3ORF1 ............................................................................................ 25 1.6.9. NDUFAF2 (B17.2L) ........................................................................... 25 1.6.10. FOXRED1 ....................................................................................... 26 1.6.11. Apoptosis Inducing Factor (AIF)...................................................... 26 1.6.12. NUBPL / IND1 / INDH ..................................................................... 27 1.6.13. MidA / NDUFAF7 ............................................................................ 28 Chapter 2…………………………………………. ................................................. 30 NOVEL COMPLEX I MUTANTS REVEALED FROM A FORWARD GENETIC SCREEN CONDUCTED IN Chlamydomonas reinhardtii ............................ 30 2.1. Introduction .................................................................................. 30 2.1.1. Chlamydomonas reinhardtii as a model system for the study of mitochondrial Complex