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Volume 272, Number 18, Issue of May 2, 1997 pp. 11750-11756
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Site-directed Mutagenesis of the Yeast V-ATPase A Subunit

(Received for publication, January 7, 1997, and in revised form, February 21, 1997)

Qing Liu , Xing-Hong Leng , Paul R. Newman , Elena Vasilyeva , Patricia M. Kane and Michael Forgac

From the Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111 and the  Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse, New York 13210

To investigate the function of residues at the catalytic nucleotide binding site of the V-ATPase, we have carried out site-directed mutagenesis of the VMA1 gene encoding the A subunit of the V-ATPase in yeast. Of the three cysteine residues that are conserved in all A subunits sequenced thus far, two (Cys²⁸⁴ and Cys⁵³⁹) appear essential for correct folding or stability of the A subunit. Mutation of the third cysteine (Cys²⁶¹), located in the glycine-rich loop, to valine, generated an enzyme that was fully active but resistant to inhibition by N-ethylmalemide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and oxidation. To test the role of disulfide bond formation in regulation of vacuolar acidification in vivo, we have also determined the effect of the C261V mutant on targeting and processing of the soluble vacuolar protein carboxypeptidase Y. No difference in carboxypeptidase Y targeting or processing is observed between the wild type and C261V mutant, suggesting that disulfide bond formation in the V-ATPase A subunit is not essential for controlling vacuolar acidification in the Golgi. In addition, fluid phase endocytosis of Lucifer Yellow, quinacrine staining of acidic intracellular compartments and cell growth are indistinguishable in the C261V and wild type cells.

Mutation of G250D in the glycine-rich loop also resulted in destabilization of the A subunit, whereas mutation of the lysine residue in this region (K263Q) gave a V-ATPase complex which showed normal levels of A subunit on the vacuolar membrane but was unstable to detergent solubilization and isolation and was totally lacking in V-ATPase activity. By contrast, mutation of the acidic residue, which has been postulated to play a direct catalytic role in the homologous F-ATPases (E286Q), had no effect on stability or assembly of the V-ATPase complex, but also led to complete loss of V-ATPase activity. The E286Q mutant showed labeling by 2-azido-[³²P]ATP that was approximately 60% of that observed for wild type, suggesting that mutation of this glutamic acid residue affected primarily ATP hydrolysis rather than nucleotide binding.

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