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The Function of the Qcr7 Protein of the Mitochondrial Ubiquinol-Cytochrome C Oxidoreductase of Saccharomyces Cerevisiae

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The Function of the Qcr7 Protein of the Mitochondrial Ubiquinol-Cytochrome C Oxidoreductase of Saccharomyces Cerevisiae The Function of the Qcr7 Protein of the Mitochondrial Ubiquinol-cytochrome c Oxidoreductase of Saccharomyces cerevisiae by Suzann Malaney A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Biochemistry University of Toronto OCopyright by Suzann Malaney 1997 National Library Bibliothèque nationale 1*1 of Canada du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 Wellington Sireet 395. me Wellington ÛtÎaiaiva ON K1A ON4 OttawaON KtAON4 canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence dowing the exclusive permettant à la National Library of Canada to Biblioîhèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de rnicrofiche/film, 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 thése. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. The Function of the Qcr7 Protein of the Mitochondrial U biquinol-cytochrome c Oxidoreductase of Saccharomyces cerevisiae Doctor of Philosophy, 1997 Suzann Malaney Department of Biochemistry, University of Toronto ABSTRACT The respiratory chain enzyme ubiquinol-cytochrome c oxidoreductase (also termed bcl complex or complex III) of Saccharomyces cerevisiae contains 1 0 subunits and resides in the inner mitochondrial membrane (IMM). This multisubunit enzyme complex is involved in the transfer of electrons from ubiquinol to cytochrome c and in the establishment of an electrochemical gradient by translocation of protons across the IMM. A previous study has shown that inactivation of the yeast nuclear gene QCR7, which encodes subunit 7 (also referred to as Qcr7 protein or 14 kDa subunit) of the bci complex leads to an inactive enzyme. The bel complex of the mutant strain lacks holocytochrome b and has reduced levels of apocytochrome b, the Rieske iron-sulfur protein (ISP), and the 11 kDa subunit. Although this study showed that the Qcr7 protein is essential for respiration, the exact role of this subunit is not known. In the present study 1 have shown by circular dichroism that an amino-terminal peptide of subunit 7 is a-helical. I have also studied the effect of mutations in the amino-terminus of--this protein on mitochondrial targeting, assembly of the bel complex, and proton translocation. Mutant proteins were analyzed by overexpression of mutated qcr7 genes in the strain YSM-qcr7A, which contains a disrupted chromosomal QCR7 gene, and by comparison to Qcr7 protein from a YSM-qcr7A strain in which the wild type QCR 7 gene is overexpressed. Respiration proficiency and the activity of the bci complex were monitored. Based on these preliminary data, strains expressing mutated proteins that lacked the N-terminal 7, 10, 14, and 20 residues (after Met-1) of subunit 7 referred to as Qcr7p-~7,-AI 0, -a14, and -a20, respectively, and strains expressing versions of Qcr7p-A7 that contained point mutations RI OK, Dl3V, and RiOI/GlZV were chosen for further study. Al1 the mutated versions of the Qcr7 protein, with the exception of Qcr7p-al0 and Qcr7p-a7(D13V), are present in the mitochondria (frorn cells grown at 300C) at reduced levels of approximately 55% when compared to Qcr7 protein from the strain overexpressing wild type QCR7. In contrast, Qcr7p-~lO is not detectable and Qcr7p-~7(013V)is present at wild type levels. This may implicate the Qcr7 protein amino-terminus in import or in conferring stability to the protein. The activity of complex III in mitochondria from strains with Qcr7p-a7 and Qcr7p-a7(Rl OK) was normal. In contrast, strains overexpressing qcr7 genes encoding Qcr7p-~lO, Qcr7p-A14, Qcr7p-~20,Qcr7p-~7(D13V), and Qcr7p- A~(R1 OVG12V) were respiration-deficient. Western blot analyses from this latter group of mutants indicate that the bcl complex in respiration-deficient cells displays a significant variation of iii reduced levels of the 11 kDa subunit, as well as combined intermediate and mature ISP and cytochrome cl. Spectrophotometric analyses indicate that the amount of holocytochrome b was reduced in the strain containing Qcr?p-~7(RlOI/G12V) and lacking in the strain with Qcr7p-d7(D13V). ATP synthesis (an indirect measure of proton translocation) in mitochondria was comparable to the wild type in al1 respiration-proficient strains tested, including the strain with Qcr7p-A7. Based on the results of this work, I concluded that the amino- terminus of the Qcr7 protein is essential for the functional assembly of ubiquinol-cytochrome c oxidoreductase. In addition to the proposed function in assernbly, the N-terminal seven residues may facilitate import into rnitochondria. DEDICATION This work is dedicated to my two little daughters Maxine and Michelle. May it be an inspiration to them throughout their lives. This work is also dedicated to my husband Robert for his love. ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. Brian H. Robinson. While in his laboratory for five years I have learned a tremendous amount about science and life. He has always been supportive, encouraging, and full of ideas. I thank him for always having an open door no matter how busy he was. I also thank him for accepting me into his laboratory and giving me the opportunity to accomplish this work. I would like to thank my CO-supervisors Dr. Charles Deber and Dr. David Williams for al1 their encouragement, support, and constructive criticism. I would also like to acknowledge contribution to the thesis by Dr. Jacqueline Segall. I am very grateful to Dr. Bernard L. Trumpower from Dartmouth Medical School from whom I have gained much knowledge over the years. I would also like to thank Dr. Peter Lewis, Dr. Reinhart Reithrneier, and Dr. Shelagh Ferguson-Miller (outside examiner from Michigan State University) for critically proofreading this manuscript and for many useful comments. Somebody who deserves special mention is my husband Robert. A scientist himself (Astrophysicist), he has been an inspiration and a hero to me from the second year of my undergraduate degree. Without him this work would have never been accomplished. His dedication as a father and a husband has made it possible for me to complete my research. I would like to thank rny fellow graduate students and CO- workers, some of whom deserve special mention. Dr. Frank Merante and Dr. Sandeep Raha were never too busy to help out, talk about science, or play practical jokes. Thank you to Tomoko Myint for her endless friendship and encouragement. I would also like to thank Dr. Sandeep Raha, and Dr. Mingfu Ling for critically proofreading this dissertation and for many useful comrnents. Thank you to Maureen Waite, our administrative assistant, for easing my work on many occasions. I would like to thank my family, especially my mother, for her never-ending support and for believing in me. Her encouragement has given me strength. 1 also thank my mother as well as my sister- in-law Linda for coming from abroad to babysit. Thank you Maxine and Michelle for brightening up even those days on which none of my experiments worked. TABLE of CONTENTS DEDIUTON............................ .. .................................................................................... v ACKNOW LEDGEM ENTS.. ................... .... ... .......................................................... .vi TABLE of CONTENTS.. ............................................................................................ .viit a.. a.* a.* UST of FIGURES ....................................................................................................... XIII LIST of TABLES............. ,... .............................................. .. ............ .. ......... m* ABBREVWONS ................................................................................................. xvii CtIAFl€R 1: Introduction................................................................................... 1 Components of the mitochondrial respiratory chain.............. ......... 1 O 1. NADH dehydrogenases....... .. ............................................................................. 1 0 II. Succinate-ubiquinone reductase............................................................... 1 1 III. Ubiquinolcytochrorne c oxidoreductase ................................................. 1 2 vii i The Rieske ironsulfur protein............................................................... 1 7 What about the supernumerary subunits of complex [Il?........... 19 The supernumerary subunits of complex III................ .. ................ 20 Subunit 6 may regulate the activity of complex III...................... 20 Subunit 7: the protein of the current study.................................... 21 Subunit 8: a ubiquinone-binding protein ? ....................................... 23 Subunit 9.......................................................................................................... 24 Subunitl O........................................................................................................ 25 The core proteins.........................................................................................
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