The proton-translocating core of eukaryotic vacuolar H+-ATPase (V-ATPase), V0 consists of a hexameric arrangement of transmembrane α-helices formed from the related polypeptides, subunit c and subunit c′′. The former is comprised of four transmembrane α-helices, whilst the latter has an extra transmembrane domain at its N-terminus. In addition, the fungal form of V0 contains a minor subunit c-related polypeptide, subunit c′. All three are required for activity of the proton pump in Saccharomyces cerevisiae. We have introduced cysteine residues in the N-terminal extension of subunit c′′ in a cysteine-free form. All mutant forms are active in the V-ATPase from S. cerevisiae. Oxidation of vacuolar membranes containing the cysteine-replaced forms gave a cross-linked product of 42000Da. Analysis of this species showed it to be a dimeric form of subunit c′′, and further studies confirmed there are two copies of subunit c′′ in the V-ATPases in which it is present. Co-expression of double cysteine-replaced forms of both subunit c and c′′ gave rise to only homotypic cross-linked forms. Also, subunit c oligomeric complexes are present in vacuolar membranes in the absence of subunit c′′, consistent with previous observations showing hexameric arrangements of subunit c in gap-junction-like membranes. In vitro studies showed subunit c′′ can bind to subunit c and itself. The extent of binding can be increased by removal of the N-terminal domain of subunit c′′. This domain may therefore function to limit the copy number of subunit c′′ in V0. A deletion study shows that the domain is essential for the activity of subunit c′′. The results can be combined into a model of V0 which contains two subunit c′′ protomers with the extra transmembrane domain located toward the central pore. Thus the predicted stoichiometry of V0 in which subunit c′′ is present is subunit c3:subunit c′1 :subunit c′2. On the basis of the mutational and binding studies, it seems likely that two copies of subunit c′′ are next to each other.
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September 2002
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Research Article|
September 15 2002
Evidence that there are two copies of subunit c″ in V0 complexes in the vacuolar H+-ATPase
Lucien C.D. GIBSON;
Lucien C.D. GIBSON
∗School of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow, G4 0BA, U.K.,
†CRC Beatson Laboratories, Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD, U.K.
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Graham CADWALLADER;
Graham CADWALLADER
∗School of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow, G4 0BA, U.K.,
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Malcolm E. FINBOW
Malcolm E. FINBOW
1
∗School of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow, G4 0BA, U.K.,
1To whom correspondence should be addressed (e-mail M.Finbow@gcal.ac.uk).
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Publisher: Portland Press Ltd
Received:
January 29 2002
Revision Received:
May 21 2002
Accepted:
May 31 2002
Accepted Manuscript online:
May 31 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 366 (3): 911–919.
Article history
Received:
January 29 2002
Revision Received:
May 21 2002
Accepted:
May 31 2002
Accepted Manuscript online:
May 31 2002
Citation
Lucien C.D. GIBSON, Graham CADWALLADER, Malcolm E. FINBOW; Evidence that there are two copies of subunit c″ in V0 complexes in the vacuolar H+-ATPase. Biochem J 15 September 2002; 366 (3): 911–919. doi: https://doi.org/10.1042/bj20020171
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