TY - JOUR
T1 - Role of transmembrane segment M8 in the biogenesis and function of yeast plasma-membrane H+-ATPase
AU - Guerra, Guadalupe
AU - Petrov, Valery V.
AU - Allen, Kenneth E.
AU - Miranda, Manuel
AU - Pardo, Juan Pablo
AU - Slayman, Carolyn W.
N1 - Funding Information:
This work was supported by research grant GM15761 from the National Institute of General Medical Sciences, grant 07-04-00419-a from the Russian Foundation for Basic Research, and grant NS-4318.2006.4 from the Support Fund of Leading Scientific Schools of Russia. The authors are grateful to Silvia Lecchi, Brett Mason, Alberto Rivetta, and Clifford Slayman for helpful discussions.
PY - 2007/10
Y1 - 2007/10
N2 - Of the four transmembrane helices (M4, M5, M6, and M8) that pack together to form the ion-binding sites of P2-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 Ca2+ binding in the Ca2+-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.
AB - Of the four transmembrane helices (M4, M5, M6, and M8) that pack together to form the ion-binding sites of P2-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 Ca2+ binding in the Ca2+-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.
UR - http://www.scopus.com/inward/record.url?scp=34948875775&partnerID=8YFLogxK
U2 - 10.1016/j.bbamem.2007.04.029
DO - 10.1016/j.bbamem.2007.04.029
M3 - Artículo
SN - 0005-2736
VL - 1768
SP - 2383
EP - 2392
JO - Biochimica et Biophysica Acta - Biomembranes
JF - Biochimica et Biophysica Acta - Biomembranes
IS - 10
ER -