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J. Biol. Chem., Vol. 278, Issue 47, 46396-46402, November 21, 2003

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Expression and Function of the Mouse V-ATPase d Subunit Isoforms^*

Tsuyoshi Nishi, Shoko Kawasaki-Nishi, and Michael Forgac

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

We have identified a cDNA encoding a novel isoform of the mouse V-ATPase d subunit (d2). The protein encoded is 350 amino acids in length and shows 42 and 67% identity to the yeast d subunit (Vma6p) and the mouse d1 isoform, respectively. Reverse transcriptase-PCR analysis using isoform-specific primers demonstrate that d2 is expressed mainly in kidney and at lower levels in heart, spleen, skeletal muscle, and testis. Although d1 and d2 show similar levels of sequence homology to Vma6p, only the d1 isoform can complement the phenotype of a yeast strain in which VMA6 has been disrupted when cells are grown at 30 °C. The d2 isoform, however, can complement the vma6 phenotype when cells are grown at 25 °C. Moreover, partial assembly of the V-ATPase complex on the vacuolar membrane can be detected under these conditions, although assembly is significantly lower than that observed for the strain expressing Vma6p. This reduced assembly is also reflected in a reduced level of concanamycin-sensitive ATPase activity and proton transport in isolated vacuoles. Comparison of the kinetic properties of V-ATPase complexes containing Vma6p and d1 demonstrate that although the K_m for ATP hydrolysis is similar (0.26 and 0.31 mM, respectively), the coupling ratio (proton transport/ATP hydrolysis) is 3–6-fold higher for d1-containing complexes than for Vma6p-containing complexes. These results suggest that subunit d may play a role in coupling of proton transport and ATP hydrolysis.

Received for publication, April 15, 2003 , and in revised form, August 22, 2003.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY145896 for the cDNA encoding the mouse d2 subunit.

^* This work was supported by National Institutes of Health Grant GM34478 (to M. F.) and E. coli strains were provided through National Institutes of Health Grant DK 34928. 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.

To whom correspondence should be addressed. Tel.: 617-636-6939; Fax: 617-636-0445; E-mail: michael.forgac{at}tufts.edu.

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