Membrane-spanning M5 and M6 segments, which play a role in the formation of cation transport sites in H(+)-, Ca2(+)-, K(+)-, Na(+)-, and other P2-ATPases, are connected by a short extracytoplasmic loop. In the yeast plasma membrane H(+)-ATPase, which belongs to a family of P2-ATPases, the loop is connected to M5 and M6 through the Asp-714 and Asp-720 residues. In this work, the effect of point amino, acidreplacements of Asp-714 and Asp-720 by Ala, Val, Asn, and Glu residues on the function of the enzyme was studied. The Asp714Asn point mutant possessed activities similar to those of the wild-type enzyme, whereas the replacement of Asp-714 by other amino acid residues disrupted biogenesis and led to a loss of activity. All mutants with substitution of Asp-720 were expressed and possessed relatively high activity. The D720V mutant displayed significantly reduced expression levels, activity, H+ transport, and ATP hydrolyzing activity. Thus, substitutions of Asp-714, except for the D714N mutant, led to significant defects in biogenesis and/or function of the enzyme. The results indicate the important role for the Asp-714 residue in biogenesis, structure stability, and enzyme function.

• PMID: 25707108
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Petrov VV, Ibragimov RI

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