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J. Biol. Chem., Vol. 281, Issue 32, 22752-22760, August 11, 2006

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The E and G Subunits of the Yeast V-ATPase Interact Tightly and Are Both Present at More Than One Copy per V₁ Complex^*

From the Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210

The E and G subunits of the yeast V-ATPase are believed to be part of the peripheral or stator stalk(s) responsible for physically and functionally linking the peripheral V₁ sector, responsible for ATP hydrolysis, to the membrane V₀ sector, containing the proton pore. The E and G subunits interact tightly and specifically, both on a far Western blot of yeast vacuolar proteins and in the yeast two-hybrid assay. Amino acids 13-79 of the E subunit are critical for the E-G two-hybrid interaction. Different tagged versions of the G subunit were expressed in a diploid cell, and affinity purification of cytosolic V₁ sectors via a FLAG-tagged G subunit resulted in copurification of a Myc-tagged G subunit, implying more than one G subunit was present in each V₁ complex. Similarly, hemagglutinin-tagged E subunit was able to affinity-purify V₁ sectors containing an untagged version of the E subunit from heterozygous diploid cells, suggesting that more than one E subunit is present. Overexpression of the subunit G results in a destabilization of subunit E similar to that seen in the complete absence of subunit G (Tomashek, J. J., Graham, L. A., Hutchins, M. U., Stevens, T. H., and Klionsky, D. J. (1997) J. Biol. Chem. 272, 26787-26793). These results are consistent with recent models showing at least two peripheral stalks connecting the V₁ and V₀ sectors of the V-ATPase and would allow both stalks to be based on an EG dimer.

Received for publication, February 14, 2006 , and in revised form, June 1, 2006.

^* This work was supported by National Institutes of Health Grant R01 GM50322 (to P. M. K.). 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 Biochemistry and Molecular Biology, State University of New York Upstate Medical University, 750 East Adams St., Syracuse, NY 13210. Tel.: 315-464-8742; Fax: 315-464-8750; E-mail: kanepm{at}upstate.edu.

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