Biogenesis and function of the yeast plasma-membrane H(+)-ATPase.
Biogenesis and function of the yeast plasma-membrane H(+)-ATPase.
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发表时间：
2000
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通讯作者：
A. Ambesi;M. Miranda;V. Petrov;C. Slayman
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作者：
A. Ambesi;M. Miranda;V. Petrov;C. Slayman
酵母质膜中最丰富的蛋白质之一是P型H（+）-ATP酶，它将质子泵出细胞，为各种H（+）依赖性协同转运蛋白提供驱动力。ATP酶是一种100 kDa的多肽，通过10个跨膜α-螺旋锚定在脂质双层中。它在结构和功能上与动物细胞的P型Na（+）、K（+）-、H（+）、K（+）-和Ca（2+）-ATP酶以及植物细胞的H（+）-ATP酶相关，并且与它们共享一种特征性反应机制，其中ATP通过共价的β-乙酰基磷酸中间体分解为ADP和无机磷酸盐（P（i））。最近获得了粗糙脉孢菌H（+）-ATP酶和动物细胞肌浆网Ca（2+）-ATP酶的8 nm分辨率的冷冻电镜图像。该分子的膜包埋部分（可能包含阳离子易位途径）通过一个狭窄的茎连接到一个大的多域细胞质部分，已知含有ATP结合和磷酸化位点。在结构研究的同时，正在努力通过定点诱变的方法来剖析几种P型ATP酶的结构/功能关系。本文综述了酵母PMA_1 H（+）-ATP酶的三种不同的突变体：（1）折叠不良并保留在内质网中的突变型ATP酶，（2）构象平衡从E（2）状态（特征为对钒酸的高亲和力）转变为E（1）状态（特征为对ATP的高亲和力）的突变型ATP酶，（3）构象平衡从E（2）状态转变为E（1）状态的突变型ATP酶，（4）构象平衡从E（2）状态转变为E（1）状态的突变型ATP酶。（3）ATP水解与质子泵作用偶联改变的突变体。虽然在完全理解转运机制之前还有很多东西需要了解，但这些突变体有助于鉴定蛋白质折叠、构象变化和H（+）：ATP偶联所需的多肽的关键部分。
One of the most abundant proteins in the yeast plasma membrane is the P-type H(+)-ATPase that pumps protons out of the cell, supplying the driving force for a wide array of H(+)-dependent cotransporters. The ATPase is a 100 kDa polypeptide, anchored in the lipid bilayer by 10 transmembrane alpha-helices. It is structurally and functionally related to the P-type Na(+),K(+)-, H(+),K(+)- and Ca(2+)-ATPases of animal cells and the H(+)-ATPases of plant cells, and it shares with them a characteristic reaction mechanism in which ATP is split to ADP and inorganic phosphate (P(i)) via a covalent beta-aspartyl phosphate intermediate. Cryoelectron microscopic images of the H(+)-ATPase of Neurospora crassa and the sarcoplasmic reticulum Ca(2+)-ATPase of animal cells have recently been obtained at 8 nm resolution. The membrane-embedded portion of the molecule, which presumably houses the cation translocation pathway, is seen to be connected via a narrow stalk to a large, multidomained cytoplasmic portion, known to contain the ATP-binding and phosphorylation sites. In parallel with the structural studies, efforts are being made to dissect structure/function relationships in several P-type ATPases by means of site-directed mutagenesis. This paper reviews three phenotypically distinct classes of mutant that have resulted from work on the yeast PMA1 H(+)-ATPase: (1) mutant ATPases that are poorly folded and retained in the endoplasmic reticulum; (2) mutants in which the conformational equilibrium has been shifted from the E(2) state, characterized by high affinity for vanadate, to the E(1) state, characterized by high affinity for ATP; and (3) mutants with altered coupling between ATP hydrolysis and proton pumping. Although much remains to be learned before the transport mechanism can be fully understood, these mutants serve to identify critical parts of the polypeptide that are required for protein folding, conformational change and H(+):ATP coupling.