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The Role of Cox20 in Cox2 Maturation and Cytochrome C Oxidase Assembly

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The Role of Cox20 in Cox2 Maturation and Cytochrome C Oxidase Assembly CORE Metadata, citation and similar papers at core.ac.uk Provided by The University of Utah: J. Willard Marriott Digital Library THE ROLE OF COX20 IN COX2 MATURATION AND CYTOCHROME C OXIDASE ASSEMBLY by Elliott Carter Ferris A thesis submitted to the faculty of The University of Utah in partial fulfillment of the requirement for the degree of Master of Science Department of Biochemistry University of Utah December 2011 Copyright © Elliott Carter Ferris 2011 All Rights Reserved The University of Utah Graduate School STATEMENT OF THESIS APPROVAL The thesis of Elliott Carter Ferris has been approved by the following supervisory committee members: Dennis R. Winge , Chair 10/27/11 Date Approved Chris Hill , Member 10/27/11 Date Approved Martin Horvath , Member 10/28/11 Date Approved and by Chris Hill , Chair of the Department of Biochemistry and by Charles A. Wight, Dean of The Graduate School. ABSTRACT Proper assembly of Cytochrome c Oxidase (CcO) is vital to mitochondrial respiration. The metallation and incorporation of CcO subunit 2 (Cox2) are important assembly steps that remain poorly understood. The Cox2 assembly factor Cox20 may provide a unique window on CcO assembly. Cox20 is known to bind Cox2 before the latter is incorporated into the larger CcO assembly in S. cerevisiae. Conservation of Cox20 in higher organisms and the lethality of its deletion in Drosophila melanogaster may suggest Cox20 plays a conserved role in CcO assembly. In an attempt to understand the nature of the hypothesized conserved role of Cox20, conserved residues within Cox20 were mutated and the resulting mutants studied. Mutation of a conserved cysteine pair impacted mitochondrial respiration, but did not block CcO assembly. The mutant Cox20 C87A forms a mixed disulfide species. Identifying the partner molecule may shed light on the role of Cox20. The present study finds Cox20, a 23 kD protein, in high molecular weight complexes that are unlikely to be homo-oligomeric. Identifying any other proteins in these complexes may shed light on the role Cox20 plays in CcO assembly. To this end, Cox20 was purified and analyzed by mass spectrometry (MS) to identify any accompanying proteins. Small quantities of the known CcO assembly factors Mss2, Coa1, and Mss51 were identified. TABLE OF CONTENTS ABSTRACT ............................................................................................................. iii Chapter 1 COX2 AND COX20 IN CCO ASSEMBLY ................................................... 1 Intoduction ..................................................................................................... 1 Cytochrome c Oxidase Assembly ................................................................... 2 Cox2 Maturation............................................................................................. 4 Cox20............................................................................................................. 4 2 THE ROLE OF COX20 in CCO ASSEMBLY ..............................................10 A Conserved Role for Cox20.........................................................................10 Characterization of the Role of Cox20 in CcO Assembly...............................11 Mutational Analysis of Cox20 .......................................................................11 Identify Components of the Cox2-Cox20 Pre-Assembly Intermediate............13 Progression of the Cox2-Cox20 Interaction....................................................15 Conclusions ...................................................................................................16 REFERENCES..............................................................................................25 CHAPTER 1 COX2 AND COX20 IN CYTOCHORME C OXIDASE ASSEMBLY Introduction Mitochondria are host to important metabolic reactions including oxidative phosphorylation (OXPHOS), which generates the bulk of cellular ATP. OXPHOS relies on the respiratory chain, a series of membrane embedded protein complexes, to couple electron transfer to the movement of H+ ions across the mitochondrial inner membrane (IM). The resulting electro-chemical gradient is harnessed by another membrane embedded complex, ATP synthase, to generate ATP. Mitochondrial diseases are marked by defects in OXPHOS and occur in 1 in 5000 humans.1 These diseases affect tissues with high energy demands such as nerves and muscles. Mitochondria contain a separate genome that encodes key mitochondrial proteins, but the bulk of mitochondrial proteins are nuclear encoded proteins that are imported from the cytosol. Nuclear encoded assembly factors are required for the complicated assembly of respiratory complexes, coordinating the incorporation of mitochondrial and nuclear encoded components as well as cofactors. Mitochondrial disease thus may arise from mutations in mitochondrial or nuclear encoded components of respiratory complexes, or nuclear encoded respiratory complex assembly factors. Autosomally inherited mitochondrial diseases are often 2 caused by defects in the assembly of respiratory complexes. The study of the complicated assembly of respiratory complexes is thus vital to understanding these mitochondrial diseases. Cytochme c Oxidase Assembly The terminal enzyme in the mitochondrial respiratory chain is cytochrome c oxidase (CcO or Complex IV). CcO catalyzes the transfer of electrons from the electron carrier cytochrome c to oxygen and uses the resulting energy to move protons across the IM. This enzyme is comprised of eleven subunits in Saccharomyces cerevisiae and thirteen in humans. Three core subunits, Cox1-3, are encoded in the mitochondrial genome while the remaining nuclear encoded subunits are imported into mitochondria. Cox1-3, play an active role in catalysis and require a number of cofactors. The maturation of the CcO holoenzyme thus requires insertion of three copper atoms and two modified heme cofactors. In the assembled enzyme, the binuclear copper A (CuA) site in the soluble domain of subunit 2 (Cox2) conveys electrons from the electron carrier cytochrome c in the inner membrane space (IMS) to a catalytic center within the membrane-integrated portion of subunit 1 (Cox1). This site relies on the heme moieties heme a and heme a3 and a second copper site (CuB) to catalyze the final step of the electron transport chain, the reduction of oxygen to water on the matrix side of the membrane. The dual genetic origin of the CcO subunits and the potential toxicity of the required cofactors demand a highly regulated assembly process involving a number of nuclear encoded assembly factors.2, 3 Defects in complex assembly are a common cause of electron transport chain related disease in humans.3 The human encephalomyelopathy, Leigh’s syndrome, for 3 example, is defined by compromised mitochondrial respiration affecting the central nervous system. Autosomally inherited mutations responsible for such disorders often affect factors important for the assembly of respiratory complexes as opposed to subunits present in the mature enzyme. Mutations in the CcO assembly factors SURF1 (Shy1 in S. cerevisiae), SCO1, and SCO2, for example, cause severe disorders including Leigh’s syndrome.4, 5 Study of Complex IV is thus now largely focused on the enigmatic assembly of the enzyme. CcO assembly likely occurs in a sequential manner.3 Cox1 is translated on mitochondrial ribosomes and inserted into the mitochondrial inner membrane (IM) as it is translated. The first definable Cox1 containing intermediate includes the assembly factors Mss51 and Cox14.6 Mss51 is lost as Cox1 progresses to Shy1 (SURF1) containing intermediates.5 The maturation of Cox1 within Shy1 containing intermediates requires the insertion of heme cofactors, the maturation of the CuB site, and the incorporation of additional subunits. Cox2 is presumably incorporated into a Cox1 containing intermediate after the heme moieties and the CuB site are introduced (in the crystal structure of bovine CcO, it 7 appears that Cox2 would block access to the CuB and heme sites). Cox2 itself contains a binuclear copper site, but whether this site is formed before or after incorporation into the larger Cox1 assembly is unclear. The importance of Cox2 maturation to human health is underscored by the disease caused in humans by mutations in the proteins SCO1 and 4, 8 SCO2; these copper chaperones function in maturing the CuA site. 4 Cox2 Maturation Cox2 is also translated on mitochondrial ribosomes recruited to the mitochondrial inner membrane.9 The N-terminus of the nascent polypeptide is inserted into the inner membrane as it exits the ribosomal tunnel by the ribosome-associated translocase Oxa1 (Figure 1A).10 Once the N-terminus is inserted into the membrane, Cox2 is bound by the membrane integrated chaperone Cox20.11 In S. cerevisiae, Cox20 is required for the proteolytic processing of an N-terminal leader peptide of Cox2 by the inner membrane peptidase complex Imp1/Imp2 (Figure 1B). The larger C-terminal domain is translocated across the IM by the Oxa1 paralog Cox18 and the assembly factor Mss2 (Figure 1C).12 Cox20 remains associated with Cox2 after proteolytic processing (Figure 1D). Cox2 is ultimately loaded with two copper atoms, presumably by the copper chaperone Sco1, and incorporated into the Cox1 subassembly. The soluble IMS copper chaperon Cox17 metallates the IM integrated chaperone Sco1, which in turn metallates Cox2.13 Whether CuA site maturation takes place before or after the Cox1 assembly incorporates Cox2 is unclear. Cox20 Cox20 is a
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