Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 283, Issue 8, Pages 4512-4519Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M707144200
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Funding
- NIDDK NIH HHS [P30 DK034928, DK34928] Funding Source: Medline
- NIGMS NIH HHS [R37 GM034478-22, R37 GM034478-21, R37 GM034478-20, R37 GM034478-23, R01 GM034478, GM 34478, R37 GM034478] Funding Source: Medline
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The vacuolar (H+) ATPases (V-ATPases) are large, multimeric proton pumps that, like the related family of F1F0 ATP synthases, employ a rotary mechanism. ATP hydrolysis by the peripheral V-1 domain drives rotation of a rotary complex (the rotor) relative to the stationary part of the enzyme (the stator), leading to proton translocation through the integral V-0 domain. One mechanism of regulating V-ATPase activity in vivo involves reversible dissociation of the V-1 and V-0 domains. Unlike the corresponding domains in F1F0, the dissociated V-1 domain does not hydrolyze ATP, and the free V0 domain does not passively conduct protons. These properties are important to avoid generation of an uncoupled ATPase activity or an unregulated proton conductance upon dissociation of the complex in vivo. Previous results (Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem. 275, 21761-21767) showed that subunit H(part of the stator) inhibits ATP hydrolysis by free V-1. To test the hypothesis that subunit H accomplishes this by bridging rotor and stator in free V-1, cysteine-mediated cross-linking studies were performed. Unique cysteine residues were introduced over the surface of subunit H from yeast by site-directed mutagenesis and used as the site of attachment of the photo-activated cross-linking reagent maleimido benzophenone. After UV-activated cross-linking, cross-linked products were identified by Western blot using subunit-specific antibodies. The results indicate that the subunit H mutant S381C shows cross-linking between subunit H and subunit F (a rotor subunit) in the free V-1 domain but not in the intact V1V0 complex. These results indicate that subunits H and F are proximal in free V-1, supporting the hypothesis that subunit H inhibits free V-1 by bridging the rotary and stator domains.
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