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Originally published In Press as doi:10.1074/jbc.M000546200 on March 9, 2000

J. Biol. Chem., Vol. 275, Issue 21, 15709-15716, May 26, 2000
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Functional Role of Charged Residues in the Transmembrane Segments of the Yeast Plasma Membrane H+-ATPase*

Valery V. PetrovDagger , Kristine P. Padmanabha, Robert K. Nakamoto§, Kenneth E. Allen, and Carolyn W. Slayman

From the Departments of Genetics and Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 05610

As defined by hydropathy analysis, the membrane-spanning segments of the yeast plasma membrane H+-ATPase contain seven negatively charged amino acids (Asp and Glu) and four positively charged amino acids (Arg and His). To explore the functional role of these residues, site-directed mutants at all 11 positions and at Glu-288, located near the cytoplasmic end of M3, have been constructed and expressed in yeast secretory vesicles. Substitutions at four of the positions (Glu-129, Glu-288, Asp-833, and Arg-857) had no significant effect on ATP hydrolysis or ATP-dependent proton pumping, substitutions at five additional positions (Arg-695, His-701, Asp-730, Asp-739, and Arg-811) led to misfolding of the ATPase and blockage at an early stage of biogenesis, and substitutions of Asp-143 allowed measurable biogenesis but nearly abolished ATP hydrolysis and proton transport. Of greatest interest were mutations of Glu-703 in M5 and Glu-803 in M8, which altered the apparent coupling between hydrolysis and transport. Three Glu-703 mutants (E703Q, E703L, E703D) showed significantly reduced pumping over a wide range of hydrolysis values and thus appeared to be partially uncoupled. At Glu-803, by contrast, one mutant (E803N) was almost completely uncoupled, while another (E803Q) pumped protons at an enhanced rate relative to the rate of ATP hydrolysis. Both Glu-703 and Glu-803 occupy positions at which amino acid substitutions have been shown to affect transport by mammalian P-ATPases. Taken together, the results provide growing evidence that residues in membrane segments 5 and 8 of the P-ATPases contribute to the cation transport pathway and that the fundamental mechanism of transport has been conserved throughout the group.

* This work was supported by Grant GM15761 from the NIGMS, National Institutes of Health (to C. W. S.) and a postdoctoral fellowship from NIGMS (to K. P. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom reprint requests should be addressed: Dept. of Genetics, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. Tel.: 203-785-2690; Fax: 203-737-1771; E-mail: cw_slaymanlab@yale.edu.

§ Present address: Dept. of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22906.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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