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J Biol Chem, Vol. 273, Issue 51, 34328-34334, December 18, 1998
From the Departments of Genetics and Cellular & Molecular
Physiology, Yale University School of Medicine,
New Haven, Connecticut 06520
The plasma-membrane H+-ATPase
of Saccharomyces cerevisiae, which belongs to the
P2 subgroup of cation-transporting ATPases, is encoded by
the PMA1 gene and functions physiologically to pump protons
out of the cell. This study has focused on hydrophobic transmembrane
segments M5 and M6 of the H+-ATPase. In particular, a
conserved aspartate residue near the middle of M6 has been found to
play a critical role in the structure and biogenesis of the ATPase.
Site-directed mutants in which Asp-730 was replaced by an uncharged
residue (Asn or Val) were abnormally sensitive to trypsin, consistent
with the idea that the proteins were poorly folded, and
immunofluorescence confocal microscopy showed them to be arrested in
the endoplasmic reticulum. Similar defects are known to occur when
either Arg-695 or His-701 in M5 is replaced by a neutral residue
(Dutra, M. B., Ambesi, A., and Slayman, C. W. (1998)
J. Biol. Chem. 273, 17411-17417). To search for
possible charge-charge interactions between Asp-730 and Arg-695 or
His-701, double mutants were constructed in which positively and
negatively charged residues were swapped or eliminated. Strikingly, two
of the double mutants (R695D/D730R and R695A/D730A) regained the
capacity for normal biogenesis and displayed near-normal rates of ATP
hydrolysis and ATP-dependent H+ pumping. These
results demonstrate that neither Arg-695 nor Asp-730 is required for
enzymatic activity or proton transport, but suggest that there is a
salt bridge between the two residues, linking M5 and M6 of the 100-kDa polypeptide.
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