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J. Biol. Chem., Vol. 282, Issue 47, 34058-34065, November 23, 2007

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Arrangement of Subunits in the Proteolipid Ring of the V-ATPase^*

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

The vacuolar ATPases (V-ATPases) are multisubunit complexes containing two domains. The V₁ domain (subunits A–H) is peripheral and carries out ATP hydrolysis. The V₀ domain (subunits a, c, c', c'', d, and e) is membrane-integral and carries out proton transport. In yeast, there are three proteolipid subunits as follows: subunit c (Vma3p), subunit c' (Vma11p), and subunit c'' (Vma16p). The proteolipid subunits form a six-membered ring containing single copies of subunits c' and c'' and four copies of subunit c. To determine the possible arrangements of proteolipid subunits in V₀ that give rise to a functional V-ATPase complex, a series of gene fusions was constructed to constrain the arrangement of pairs of subunits in the ring. Fusions containing c'' employed a truncated version of this protein lacking the first putative transmembrane helix (which we have shown previously to be functional), to ensure that the N and C termini of all subunits were located on the luminal side of the membrane. Fusion constructs were expressed in strains disrupted in c', c'', or both but containing a wild copy of c to ensure the presence of the required number of copies of subunit c. The c-c''(TM1), c''(TM1)-c', and c'-c constructs all complemented the vma^– phenotype and gave rise to complexes possessing greater than 25% of wild-type levels of activity. By contrast, neither the c-c', the c'-c''(TM1), nor the c''(TM1)-c constructs complemented the vma^– phenotype. These results suggest that functionally assembled V-ATPase complexes contain the proteolipid subunits arranged in a unique order in the ring.

Received for publication, May 25, 2007 , and in revised form, August 30, 2007.

^* This work was supported by National Institutes of Health Grant GM34478 (to M. F.), a postdoctoral fellowship from the NE Affiliate of the American Heart Association (to Y. W.), and a postdoctoral fellowship from the Canadian Institutes of Health Research (to D. J. C.). 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.

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