Cytochrome c Oxidase-Lipid Interface from the Protein Side

Georgevich, Gradimir; Ra, Chisei

doi:10.1016/s0006-3495(82)84602-x

Cited by 15 publications

(4 citation statements)

References 2 publications

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“…The use of hydrophobic reagents, such as adamantane diazirine [38] and arylazidophospholipids [39,401, has been of invaluable help in mapping subunit 111. These reagents are lipophilic and thus react with bilayer-intercalated parts of the most hydrophobic subunits, including subunit 111.…”

Section: Chemical Modification Ojsuhunit 111mentioning

confidence: 99%

Cytochrome-c oxidase. Subunit structure and proton pumping

Brunori¹,

et al. 1987

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Section: Chemical Modification Ojsuhunit 111mentioning

confidence: 99%

Cytochrome-c oxidase. Subunit structure and proton pumping

Brunori¹,

et al. 1987

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“…The arrangement of protein with respect to the lipid bilayer has also been examined by labeling with radioactive arylazidophospholipids (Bisson et al, 1979;Prochaska et al, 1980), iodonaphthyl azide (Cerletti & Schatz, 1979), and adamantane diazirine (Georgevich & Capaldi, 1982). Subunits I and III were heavily labeled by each of these hydrophobic reagents and must contribute the major portion of the protein in contact with lipid.…”

mentioning

confidence: 99%

Arrangement of subunit IV in beef heart cytochrome c oxidase probed by chemical labeling and protease digestion experiments

Malatesta¹,

Darley‐Usmar²,

Jong³

et al. 1983

Biochemistry

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The arrangement of subunit IV in beef heart cytochrome c oxidase has been explored by chemical labeling and protease digestion studies. This subunit has been purified from four samples of cytochrome c oxidase that had been reacted with N-(4-azido-2-nitrophenyl)-2-aminoethyl[35S]-sulfonate (NAP-taurine), diazobenzene[35S]sulfonate, 1-myristoyl-2-[12-[(4-azido-2-nitrophenyl)amino]lauroyl]-sn-glycero-3- [14C]phosphocholine (I), and 1-palmitoyl-2-(2-azido-4-nitrobenzoyl)-sn-glycero-3-[3H]phosphocholine (II), respectively. The labeled polypeptide was then fragmented by cyanogen bromide, at arginyl side chains with trypsin (after maleylation), and the distribution of the labeling within the sequence was analyzed. The N-terminal part of subunit IV (residues 1-71) was shown to be heavily labeled by water-soluble, lipid-insoluble reagents but not by the phospholipid derivatives. These latter reagents labeled only in the region of residues 62-122, containing the long hydrophobic and putative membrane-spanning stretch. Trypsin cleavage of native cytochrome c oxidase complex at pH 8.2 was shown to clip the first seven amino acids from subunit IV. This cleavage was found to occur in submitochondrial particles but not in mitochondria or mitoplasts. These results are interpreted to show that subunit IV is oriented with its N terminus on the matrix side of the mitochondrial inner membrane and spans the membrane with the extended sequence of hydrophobic lipid residues 79-98 buried in the bilayer.

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“…This polypeptide is very hydrophilic, without the long stretches of hydrophobic residues that characterize the majority of transmembrane polypeptides [see Capaldi (1990a)]. Moreover, this subunit is not labeled by any of the bilayer-intercalated reagents that have been used to date (Bisson et al, 1979;Georgevich & Capaldi, 1982;, making a transmembrane arrangement unlikely. Subunit Va is cross-linked to subunit II in relatively high yield by DSP, a reagent that reacts with lysine residues (Briggs & Capaldi, 1977; Jarausch & Kadenbach, 1985a).…”

Section: Discussionmentioning

confidence: 99%

Topology of subunits of the mammalian cytochrome c oxidase: relationship to the assembly of the enzyme complex

Zhang¹,

Ewart²,

Capaldi³

1991

Biochemistry

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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.

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Cytochrome c Oxidase-Lipid Interface from the Protein Side

Cited by 15 publications

References 2 publications

Cytochrome-c oxidase. Subunit structure and proton pumping

Cytochrome-c oxidase. Subunit structure and proton pumping

Arrangement of subunit IV in beef heart cytochrome c oxidase probed by chemical labeling and protease digestion experiments

Topology of subunits of the mammalian cytochrome c oxidase: relationship to the assembly of the enzyme complex

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