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J. Biol. Chem., Vol. 283, Issue 8, 4512-4519, February 22, 2008

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Subunit H of the Vacuolar (H⁺) ATPase Inhibits ATP Hydrolysis by the Free V₁ Domain by Interaction with the Rotary Subunit F^*

From the Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111

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 V₁ 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 V₀ domain. One mechanism of regulating V-ATPase activity in vivo involves reversible dissociation of the V₁ and V₀ domains. Unlike the corresponding domains in F₁F₀, the dissociated V₁ 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 V₁. To test the hypothesis that subunit H accomplishes this by bridging rotor and stator in free V₁, 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 V₁ domain but not in the intact V₁V₀ complex. These results indicate that subunits H and F are proximal in free V₁, supporting the hypothesis that subunit H inhibits free V₁ by bridging the rotary and stator domains.

Received for publication, August 27, 2007 , and in revised form, December 6, 2007.

^* This work was supported in part by National Institutes of Health Grant GM 34478 (to M. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by National Institutes of Health Training Grant DK07542.

² To whom correspondence should be addressed: Dept. of Physiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6939; Fax: 617-636-0445; E-mail: michael.forgac{at}tufts.edu.

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