Topology of subunits of the mammalian cytochrome c oxidase: relationship to the assembly of the enzyme complex.
The arrangement of three subunits of beef heart cytochrome c oxidase, subunits Va, VIa, and VIII, has been explored by chemical labeling and protease digestion studies. Subunit Va is an extrinsic protein located on the C side of the mitochondrial inner membrane. This subunit was found to label with N-(4-azido-2-nitrophenyl)-2-aminoethane[35S]sulfonate and sodium methyl 4-[3H]formylphenyl phosphate in reconstituted vesicles in which 90% of cytochrome c oxidase complexes were oriented with the C domain outermost. Subunit VIa was cleaved by trypsin both in these reconstituted vesicles and in submitochondrial particles, indicating a transmembrane orientation. The epitope for a monoclonal antibody (mAb) to subunit VIa was lost or destroyed when cleavage occurred in reconstituted vesicles. This epitope was localized to the C-terminal part of the subunit by antibody binding to a fusion protein consisting of glutathione S-transferase (G-ST) and the C-terminal amino acids 55-85 of subunit VIa. No antibody binding was obtained with a fusion protein containing G-ST and the N-terminal amino acids 1-55. The mAb reaction orients subunit VIa with its C-terminus in the C domain. Subunit VIII was cleaved by trypsin in submitochondrial particles but not in reconstituted vesicles. N-Terminal sequencing of the subunit VIII cleavage product from submitochondrial particles gave the same sequence as the untreated subunit, i.e., ITA, indicating that it is the C-terminus which is cleaved from the M side. Subunits Va and VIII each contain N-terminal extensions or leader sequences in the precursor polypeptides; subunit VIa is made without an N-terminal extension.
Zhang
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YZ
Institute of Molecular Biology, University of Oregon, Eugene 97405.
Ewart
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Capaldi
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002417:Cattle; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000276:immunology; D000939:Epitopes; D006868:Hydrolysis; D007202:Indicators and Reagents / Q000378:metabolism; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D009943:Organophosphorus Compounds / Q000378:metabolism; D011487:Protein Conformation; D013367:Submitochondrial Particles / Q000187:drug effects / Q000201:enzymology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007202:Indicators and Reagents; D009943:Organophosphorus Compounds; C054837:methyl 4-formylphenyl phosphate; D003576:Electron Transport Complex IV