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The Green Alga Chlamydomonas Reinhardtii: a New Model System To THE GREEN ALGA CHLAMYDOMONAS REINHARDTII: A NEW MODEL SYSTEM TO UNRAVEL THE ASSEMBLY PROCESS OF RESPIRATORY COMPLEXES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By María del Rosario Barbieri Graduate Program in Plant Cellular and Molecular Biology The Ohio State University 2010 Dissertation Committee Professor Patrice P. Hamel, Adviser Professor Iris Meier Professor Erich Grotewold Professor Juan D. Alfonzo ABSTRACT The general purpose of this research is to contribute to a better understanding of the mitochondrial NADH: ubiquinone oxidoreductase (Complex I) and to illustrate the current view of its assembly process. I propose to use the green alga Chlamydomonas reinhardtii as a novel model system to carry out the molecular dissection of Complex I assembly. The main objective is to discover novel genes controlling the assembly process of this multimeric enzyme. Several reasons including patient death at young age, strict regulations, and ethical concerns make the study of Complex I on humans highly difficult. Although traditionally well established model systems such as the fungi Yarrowia lipolytica and Neurospora crassa and human cell lines have proven useful to understand the role of specific subunits in the assembly process, forward genetic approaches leading to discovery of novel assembly factors have been limited by the lack of straightforward screening methodologies (REMACLE et al. 2008). The process of plant Complex I assembly is largely obscure at present, despite the progress achieved in recent years. In plants, similarly to other eukaryotes, there is no simple strategy to approach the issue of assembly. Thus, the development of a model system where both mitochondrial and nuclear genomes can be manipulated is highly desirable for the study of Complex I. ii Chapter 2 shows the use of Chlamydomonas reinhardtii as a forward genetics tool to screen for Complex I deficiency mutants and the finding of six new loci that result in defects of assembly. Based on large level of conservation of Complex I subunits in eukaryotes as well as the presence of homologs of currently proposed assembly factors, this work follows the hypothesis that the assembly process is also conserved among eukaryotes. Chapter 3 refers to the molecular identification of one of these genes as the Complex I subunit NUOB10, homolog of the bovine PSDW subunit. Also, a molecular mapping approach for another mutant suggests that the mutation does not affect a structural subunit of Complex I and that the final identification of this gene will lead to the discovery of a novel assembly factor. Finally, Chapter 4 provides an initial evaluation for the overall functional conservation of two known assembly factors: CIA30 and OXA1, both of which have a ortholog gene in Chlamydomonas. iii To my love iv ACKNOWDLEGMENTS I could not have done this work without the help and guidance of my adviser, Dr. Patrice P. Hamel. I am always grateful to him. He has been the most wonderful scientific mentor and friend through these years. In the most enduring times he has shown me the attitude to celebrate everyday achievements and overcome the obstacles of research. I would not have arrived here without his sharing of scientific thoughts with a warm cup of tea (I should also extend my gratitude to Birgit Alber, our scientific tea chat sponsor). Yet I would not be here without the support of my committee members, Dr. Iris Meier, Dr. Eric Grotewold and Dr. Juan Alfonzo. I would like to thank them for their continuous encouragement and advice. Not in my committee but an advisor nonetheless, Dr. Claire Remacle from the University of Liege, Belgium, thank you for providing your insights to my research. Two past labmates have specially contributed to this project Cécile Nouet and Véronique Larosa and deserve a special note for their technical, intellectual and spiritual contributions to this project. Thanks to my labmates through these years, Nitya, Sara Cline, Mohamed Karamoko, Stéphane Gabilly, Darren Murrey and Vincent Corvest, for their encouragement and help. v I am especially thankful to my fifth-floor Aronoff friends, especially Srilakshmi Makkena, Sowmya Venkatakrishnan, Nitya again and Sachin Teotia. They have been just wonderful, encouraging, helpful and supportive. Also, thanks to all the staff members of our department, with especial thanks to Rene Madsen, for making my life easier every day. To my friends in science, in life and in poetry, you’ve been here with me all these years, day and night, next block or next hemisphere, I am indebted to you. My dearest Fernando “Mordi”, we have not yet written our last verse. Susana, Vanessa, Tom, Elisa, Cécile, Niro, thanks for being my family in Columbus, and for the laughs and the dinners and the encouragement in the difficult times. Meye, Verónica, and Carla, you have built a piece of home far from home. Mariana L., Silvita, LauKa, Ceci O., Claudio and Kari, Flavia, you confirmed to me that we can make distance a meaningless word. My last and most special thanks are to my love, Pierre whose unconditional support and company have given me the necessary strength for this journey. To my mom, my sister Mili and brother Salva, my nephews Luli and Rodri, who understood and accepted that I needed to look for my own path. vi VITA 1974 ...............................................................Born-Buenos Aires, Argentina 2001 ...............................................................Licenciado en Ciencias Biologicas, University of Buenos Aires-Argentina 2003 to present ..............................................Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS Research Publication 1. C. Remacle, S. Cline, L. Boutaffala, S. Gabilly, V. Larosa, M.R. Barbieri, N. Coosemans, P.P. Hamel (2009). The ARG9 gene encodes the plastid resident n-1 acetyl ornithine aminotransferase 2 in the green alga Chlamydomonas reinhardtii. Eukaryotic Cell, 8 (9): 1460-1463. 2. C. Remacle, M. R. Barbieri, P. Cardol and P.P. Hamel (2008). Eukaryotic complex I: functional diversity and experimental systems to unravel the assembly process. Mol Genet Genomics, 280 (2): 93-110. 3. C. Tami, A. Peralta, R. Barbieri, A. Berinstein, E. Carrillo, O. Taboga (2004). Immunological properties of FMDV-gP64 fusion proteins expressed on SF9 cell and baculovirus surfaces. Vaccine, 23: 840-845. vii 4. M.R.Barbieri, N.L. Kerber, N.L. Pucheu, M.H. Tadros, A.F. Garcia (2002). Effect of Light and oxygen and adaptation to changing light conditions in a photosynthetic mutant in which the LHI complex of Rhv. sulfidophilum was heterologously expressed in a strain of Rb. capsulatus whose puc operon was deleted. Current Microbiology, 45: 209-216. FIELDS OF STUDY Major Field: Plant Cellular and Molecular Biology viii TABLE OF CONTENTS Page Abstract ............................................................................................................................. ii Acknowldegments................................................................................................................v Vita ........................................................................................................................... vii Publications ....................................................................................................................... vii Fields of study .................................................................................................................. viii Table of contents ................................................................................................................ ix List of tables ...................................................................................................................... xii List of figures ................................................................................................................... xiii Chapters: 1. Introduction .............................................................................................................16 1.1. Mitochondrial respiratory chain ....................................................................16 1.2. Mitochondrial Complex I ..............................................................................19 1.2.1. Complex I structure and mechanism ..................................................20 1.2.2. The assembly of Complex I ................................................................27 1.2.2.1. Core subunit mutation effects on Complex I activity and assembly ................................................................................29 1.2.2.2. Role of the accessory subunits in Complex I assembly ........31 1.2.2.3. Complex I assembly factors ..................................................32 1.3. Complex I function in plants .........................................................................38 1.3.1. Carbonic anhydrases in plant Complex I ............................................39 1.3.2. Metabolic aspects of Complex I activity defects ................................41 1.3.3. Complex I defects and effects in fertility ...........................................46 2. Dissection of Complex I assembly in the green alga Chlamydomonas reinhardtii ................................................................................................................51 ix 2.1. Introduction ...................................................................................................51
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