Role of transmembrane segment M8 in the biogenesis and function of yeast plasma-membrane H+-ATPase
Role of transmembrane segment M8 in the biogenesis and function of yeast plasma-membrane H+-ATPase
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DOI：
10.1016/j.bbamem.2007.04.029
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发表时间：
2007-10-01
影响因子：
3.4
通讯作者：
Slayman, Carolyn W.
中科院分区：
文献类型：
--
作者：
Guerra, Guadalupe;Petrov, Valery V.;Slayman, Carolyn W.
四个跨膜螺旋（M4, M5， M6和M8）聚集在一起形成p -2型atp酶的离子结合位点，其中M8迄今为止受到的关注最少。本研究利用丙氨酸扫描诱变技术绘制了酵母质膜H+- atp酶M8的结构-功能关系。突变形式的ATP酶在分泌囊泡和质膜上表达，用于测量ATP水解和ATP依赖的H+泵送。在分泌囊泡中，靠近M8胞外质端4个位置的Ala取代导致H+运输部分脱离ATP水解，而Ser-800（靠近M8中间）被Ala取代增加了H+运输的表观化学计量。在Gln取代Glu-803后，先前也有类似的增加（Petrov， V. et al.， J. Biol）。化学，275:15709-15718,2000)在已知直接促进肌浆网Ca2+- atp酶Ca2+结合的位置（Toyoshima， C.等，Nature 405: 647-655, 2000）。另外四个M8突变干扰了H+- atp酶折叠和转运到质膜；基于同源性建模，它们占据了M8与M5、M6、M7和M10正确捆绑的重要位置。综上所述，这些结果表明M8在H+- atp酶的生物发生、稳定性和生理功能中起着关键作用。(C) 2007 Elsevier B.V.版权所有
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 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. (C) 2007 Elsevier B.V. All rights reserved.