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MLRQ Subunit of NADH:Ubiquinone Oxidoreductase in the Human Mitochondrial Respiratory Chain

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The Molecular and Biochernical Characterization of the MLRQ Subunit of NADH:Ubiquinone Oxidoreductase in the Human Mitochondrial Respiratory Chain Dhush y Kanagarajah A Thesis submitted in codormity with the requirements for the degree of Master of Science Graduate Department of Biochemistry University of Toronto " Copyright by Dhushy Kanagarajah. 200 1 National Library Bibliothèque nationale I*l of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. rue Wellington Ottawa ON K1A ON4 ûttawa ON K1A ON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence ailowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distriie or seil reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/fh, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or othenirise de celle-ci ne doivent être imprimés reproduced without the author' s ou autrement reproduits sans son permission. autorisation. The Molecular and Biochemical Characterization of the MLRQ Subunit of NADH:Ubiquinone Oxidoreductase in the Humsn Mitochondrîal Respiratory Chain Master of Science, 200 1 Dhushy Kanagarajah Department of Biochemistry University of Toronto Abstract Isolated deficiency of NADH:ubiquinone oxidoreductase (Complex 1), the first enzyme of the mitochondrial respiratory chah is the most comrnon cause of human mitochondriocytopathies. In order to characterize the nuclear genes contributing to this disease, the cDNA and genomic sequences encoding the MLRQ subunit of complex I were determined. The NDUF.44 gene encoding MLRQ was localized to chromosome 7 p2 1-22 and a pseudogene was found on chromosome 1 p2 1. Tissue specific expression of MLRQ at both mRNA and protein levels was examined. Overexpression of this subunit in a patient exhibiting complex 1 deficiency is dso discussed. Extraction. immunoprecipitation and cross-linking studies revealed that while the N-teminus of MLRQ has a great afinity for phospholipids of the inner mitochondriai membrane. it likely also associates with the MWFE subunit through other intemediary subunit(s). forming part of the bulky staik region that bridges the two arms of complex 1. Acknowledgements First and foremost, 1wish to express my heartfelt thanks and gratitude to Dr. Brian Robinson for his guidance and support during the course of this degree. 1 tnily feel privileged to have had the opportunity of working with such a great supervisor. 1 am also gratefùl to my CO-supervisors,Dr. B. Sarkar and Dr. R. Baker for their theand assistance with this project. Behind every graduate student's thesis lies a support system so precious that failing to acknowledge it would be unpardonable. 1 count myself lucky to have had the privilege of working with al1 my colleagues @ast and present) at the Robinson lab, but Imust especially thank Agnieszka, Jessie, Maryanna, Nevi, So-Young and Tomoko. Their kindness and fiiendship will never be forgotten. 1 am forever indebted to Dr. Sandy Raha for al1 his expertise, encouragement and humour without which 1 would never have survived. A very special thanks goes to Maureen Waite for her friendship and for always making the time to lend me a hand. This thesis would not have been completed without the extraordinary love and support of each and every person whom 1 cal1 family. My most ardent supporters. they have been with me throughout the trials and tribulations of graduate life. For this, 1 would like to thank my uncle Milroy, my brother Dhilip and especially my husband Ramana for his patience and understanding during these past few years. Finally but most importantly. 1 would like to express my gratitude to my parents for impressing upon me the principles of hard work, perseverance and a firm belief in the merits of education. Their love and prayers have been instrumental in the completion of this study. I dedicate this thesis to the memory of my loved ones whose blessings and encouragement stiil spur me on to greater accomplishments. a-. III CONTRIBUTIONS TO THESIS Screening of PACNAC libraries and FISH Mapping: The Toronto Centre For Applied Genomics Tissue mitochondria: Dr. Sandeep Raha Table of Contents Abstract Acknowledgements Contributions to thesis Table of contents i v a.. a.. List of figures Vtlt List of tables Abbreviations Glossary of Medical Terms Chapter 1 Introduction and Objectives The mitochondrion: structure, function, mode of inheritance and associated diseases Oventiew Part 1. Mitochondrial structure. function and inheritance The mitoc hondrion Mitochondrial ultrastructure Mitochondrial DNA organization Mitochondrial replication. transcription and translation Mitochondrial protein import Energy metabolism Part II. The mitochondrial respiratory chah complexes Organization of the OXPHOS system The OXPHOS system: Role in electron transport and proton translocation Complex 1: The NADHxbiquinone oxidoreductase complex Evolution of compler 1 Subunit composition of the NADH:ubiquinone oxidoreductase corn plex (i) The flavoprotein fraction (FP) (ii) The iron-sulphur protein fraction (IP) (iii) nie hydrophobie protein fraction (HP) a) The rnitochondrially encoded subunits b) The nuclear encoded subunits Structural mode1 of complex 1 (i) Assembly of Complex 1 (ii) Spatial Organization and Subunit Interaction in Human Complex 1 32 Energy conversion in cornplex 1 35 (i) Iron-sulphur clusters. flavin and semiquinones 35 (ii) Electron transfer in cornplex 1 37 (iii) Models for coupling electron flow with proton translocation 42 Complex 1 inhibitors 47 Complex II: The Succinate-ubiquinone oxidoreductase cornplex 48 Complex III: The Ubiquinol-femcytochrome c oxidoreductase complex 49 Complex IV: The cytochrome c oxidase complex 5 1 Cornplex V: The ATP synthase complex 52 Part M. Mitochondrial disorders 54 Typical symptoms of defects in energy metabolism Mitochondrial respiratory chain diseases (i) MtDNA associated diseases (ii) Nuclear DNA associated diseases (iii) Mitochondrial respiratory chain disorders associated with neurological diseases Human Complex I deficiencies (i) MtDNA encoded defects in complex 1 (ii) Nuclear DNA encoded defects in complex 1 (iii) Free radical generation and complex 1 deficiency Objectives and Rationale 62 Chapter 2 Cloning, molecular characterization and chromosomal localization of the MLRQ subunit of hurnan NADH:ubiquinone oridoreductase Abstract Introduction Materiais and methods Part 1. Molecular Characterization of MLRQ cDNA in various tissues. cells and patient cell lines Tissue culture of cardiomyocytes and fibroblasts RNA isolation from tissues and cells cDNA synthesis and PCR cDNA cloning and sequencing of MLRQ Mutational screening of complex 1 deficient patients Part II. Genomic characterization and Iocalization of the NDUE4-C (MLRQ) gene and pseudogene Screening the PAC library Southem blot analysis of PAC clones Northem analysis of MLRQ expression Chromosomal localization Amplification. cloning and sequencing of MLRQ from genomic and PAC DNA Y AC library screening Results and discussion Part 1. Isolation and characterization of MLRQ cDNA MLRQ cDNA structure Mutational analysis of MLRQ cDNA in complex 1 deficient patients Part II. Chromosomal localization and characterization of the iVDUE4-I gene and pseudogene Library screening and FISH mapping MLRQ expression at the transcriptional level Amplification of the iVDUFA-I gene fiom genomic DNA Southem blot analysis Genomic organization of NDUFA-l The pseudogene on chromosome 1 Other MLRQ-like sequenccs in the genorne Chapter 3 Biochemical characterization, protein expression and immunoprecipitation studies pertaining to 1MLRQ and related complex 1 subunits Abstract Introduction Materials and methods Part 1. MLRQ expression in hurnan tissues and cells Antibody generation Western blot analysis of MLRQ expression Part II. Bacterial expression of MLRQ protein Design of MLRQ- fusion protein construct Induction and puritkation of MLRQ-GST fusion protein Factor Xa cleavage of fusion protein Part III. Anti-srnse expression of MLRQ in marnmalian celis Design of sense and anti-sense oriented pREP9 constructs Optimization of transfection conditions Transfection and selection with pREP9 Transfection and selection with the linearized vector pCDNA 3.l+ Western blot analysis of sense anti-sense expression in transfected cells Amplification of MLRQ From transfected cells Part IV. Association of MLRQ with other complex 1 subunits Solubilization of beef heart mitochondria Immunoprecipitation of MLRQ, MWFE and 49 kD subunits Cross-linking with DST and EGS Imrnunoprecipitation of cross-linked bovine hem mitochondria SDS-PAGE and western blot analysis of MLRQ during extraction. immunoprecipitation and cross-linking Results and discussion Part 1. Determining MLRQ fom. îûnction and expression Tissue expression of MLRQ Bacterial expression of MLRQ: Attempts at defining subunit structure vii Antisense expression of MLRQ: Attempts to detemine subunit function 1 13 Part II. Subunit interactions of MLRQ within complex 1 117 Detergent solubilization of cornplex 1 subunits 117 Proximity and association of MLRQ
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