Role of transmembrane segment M8 in the biogenesis and function of yeast plasma-membrane H(+)-ATPase.
Of the four transmembrane helices (M4, M5, M6, and M8) that pack together to form the ion-binding sites of P(2)-type ATPases, M8 has until now received the least attention. The present study has used alanine-scanning mutagenesis to map structure-function relationships throughout M8 of the yeast plasma-membrane H(+)-ATPase. Mutant forms of the ATPase were expressed in secretory vesicles and at the plasma membrane for measurements of ATP hydrolysis and ATP-dependent H(+) pumping. In secretory vesicles, Ala substitutions at a cluster of four positions near the extracytoplasmic end of M8 led to partial uncoupling of H(+) transport from ATP hydrolysis, while substitution of Ser-800 (close to the middle of M8) by Ala increased the apparent stoichiometry of H(+) transport. A similar increase has previously been reported following the substitution of Glu-803 by Gln (Petrov, V. et al., J. Biol. Chem. 275:15709-15718, 2000) at a position known to contribute directly to Ca(2+) binding in the Ca(2+)-ATPase of sarcoplasmic reticulum (Toyoshima, C., et al., Nature 405: 647-655, 2000). Four other mutations in M8 interfered with H(+)-ATPase folding and trafficking to the plasma membrane; based on homology modeling, they occupy positions that appear important for the proper bundling of M8 with M5, M6, M7, and M10. Taken together, these results point to a key role for M8 in the biogenesis, stability, and physiological functioning of the H(+)-ATPase.
Guerra
Guadalupe
G
Departmento de Microbiologia, Escuela Nacional de Ciencias Biologicas, I.P.N., Mexico, D.F., Mexico.
Petrov
Valery V
VV
Allen
Kenneth E
KE
Miranda
Manuel
M
Pardo
Juan Pablo
JP
Slayman
Carolyn W
CW
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000502:physiology; D013329:Structure-Activity Relationship
D029701:Saccharomyces cerevisiae Proteins; D006180:Proton-Translocating ATPases