title: Characterization of assembly intermediates containing subunit 1 of yeast cytochrome oxidase.
abstract: Mitochondrial-encoded Cox1p, one of the three core subunits of yeast cytochrome oxidase (COX), was previously shown to associate with regulatory proteins and nuclear-encoded subunits into five high molecular weight complexes that were proposed to constitute the pathway for biogenesis of the Cox1p assembly module. One of the intermediates (D5) was inferred, but not directly shown to exist. In the present study mitochondria of strains expressing C-terminal-tagged subunits of COX that had not been looked at previously were pulse-labeled and analyzed for the presence of newly translated Cox1p in the immunoprecipitates. These studies revealed that of the eight nuclear-encoded COX subunits, only Cox5ap, Cox6p, and Cox8p are present in the Cox1p module. Both Cox5ap and Cox8p share interfaces with Cox1p in the holoenzyme, whereas Cox6p interacts indirectly through Cox5ap. These results suggest that the subunit contacts in the holoenzyme are probably established during biogenesis of the Cox1p module. To confirm the existence of the largest Cox1p intermediates (D5), which was only inferred previously, radiolabeled Cox1p with a C-terminal tag was expressed in COX-deficient pet111 and pet494 mutants. Pulldown assays confirmed the presence of newly translated Cox1p in D5, which in wild type cannot be demonstrated directly because of its co-migration with COX in the native electrophoresis system used to separate the intermediates. Jointly, the results of these analyses substantiate our previous proposal that COX is assembled from separate assembly modules, each containing one of the mitochondrial-translated core subunits in association with a unique set of nuclear-encoded subunits.
authors.affiliation: From the Department of Biological Sciences, Columbia University, New York, New York 10027.
authors.forename: Gavin P
authors.initials: GP
authors.lastname: McStay
authors.forename: Chen-Hsien
authors.initials: CH
authors.lastname: Su
authors.forename: Susan M
authors.initials: SM
authors.lastname: Thomas
authors.forename: Jonathan Tong
authors.initials: JT
authors.lastname: Xu
authors.forename: Alexander
authors.initials: A
authors.lastname: Tzagoloff
mesh_terms: D000483:Alleles; D002467:Cell Nucleus / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D020035:Holoenzymes / Q000737:chemistry; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D020035:Holoenzymes; D029701:Saccharomyces cerevisiae Proteins; C526453:Cox1 protein, S cerevisiae; D003576:Electron Transport Complex IV
keywords: Biogenesis; Cox1p Intermediates; Cytochrome Oxidase; Membrane Biogenesis; Mitochondria; Respiratory Chain; Saccharomyces cerevisiae; Yeast Genetics
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