HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 28, 19274-19282, July 11, 2008

This Article Full Text Full Text (PDF) All Versions of this Article: 283/28/19274    most recent M802442200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Qi, J. Articles by Forgac, M. Search for Related Content PubMed PubMed Citation Articles by Qi, J. Articles by Forgac, M. Social Bookmarking                      What's this?

Function and Subunit Interactions of the N-terminal Domain of Subunit a (Vph1p) of the Yeast V-ATPase^*

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

The vacuolar (H⁺)-ATPases (V-ATPases) are ATP-dependent proton pumps that operate by a rotary mechanism in which ATP hydrolysis drives rotation of a ring of proteolipid subunits relative to subunit a within the integral V₀ domain. In vivo dissociation of the V-ATPase (an important regulatory mechanism) generates a V₀ domain that does not passively conduct protons. EM analysis indicates that the N-terminal domain of subunit a approaches the rotary subunits in free V₀, suggesting a possible mechanism of silencing passive proton transport. To test the hypothesis that the N-terminal domain inhibits passive proton flux by preventing rotation of the proteolipid ring in free V₀, factor Xa cleavage sites were introduced between the N- and C-terminal domains of subunit a (the Vph1p isoform in yeast) to allow its removal in vitro after isolation of vacuolar membranes. The mutant Vph1p gave rise to a partially uncoupled V-ATPase complex. Cleavage with factor Xa led to further loss of coupling of proton transport and ATP hydrolysis. Removal of the N-terminal domain by cleavage with factor Xa and treatment with KNO₃ and MgATP did not, however, lead to an increase in passive proton conductance by free V₀, suggesting that removal of the N-terminal domain is not sufficient to facilitate passive proton conductance through V₀. Photoactivated cross-linking using the cysteine reagent maleimido benzophenone and single cysteine mutants of subunit a demonstrated the proximity of specific sites within the N-terminal domain and subunits E and G of the peripheral stalk. These results suggest that a localized region of the N-terminal domain (residues 347–369) is important in anchoring the peripheral stator in V₁V₀.

Received for publication, March 28, 2008 , and in revised form, May 9, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant GM34478 (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 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.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				B. Ediger, S. D. Melman, D. L. Pappas Jr., M. Finch, J. Applen, and K. J. Parra The Tether Connecting Cytosolic (N Terminus) and Membrane (C Terminus) Domains of Yeast V-ATPase Subunit a (Vph1) Is Required for Assembly of V0 Subunit d J. Biol. Chem., July 17, 2009; 284(29): 19522 - 19532. [Abstract] [Full Text] [PDF]

				H. Diab, M. Ohira, M. Liu, E. Cobb, and P. M. Kane Subunit Interactions and Requirements for Inhibition of the Yeast V1-ATPase J. Biol. Chem., May 15, 2009; 284(20): 13316 - 13325. [Abstract] [Full Text] [PDF]