Atomic model for the membrane-embedded VO motor of a eukaryotic V-ATPase
Atomic model for the membrane-embedded VO motor of a eukaryotic V-ATPase
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DOI：
10.1038/nature19828
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发表时间：
2016-11-03
期刊：
影响因子：
64.8
通讯作者：
Rubinstein, John L.
中科院分区：
文献类型：
--
作者：
Mazhab-Jafari, Mohammad T.;Rohou, Alexis;Rubinstein, John L.
液泡型ATPase(V-ATPase)是由ATP驱动的质子泵，参与内吞、溶酶体降解、二次转运、TOR信号转导、破骨细胞和肾功能等过程。可溶性催化V-1区的ATP水解通过转子亚复合体的旋转推动质子通过膜包埋的V-O区转运。完整酶结构的可变性阻碍了为任何旋转ATPase的膜嵌入电机构建原子模型(1-5)。我们通过饥饿酿酒酵母(6，7)诱导了V-ATPase的V1和VO区域的解离和自动抑制，使我们能够获得VO复合体的分辨率电子冷冻显微镜图，并为其大部分亚基建立原子模型。分析揭示了ac(8)c‘c’de亚基的结构，以及我们发现并建议为一个新的亚基(f亚基)的蛋白质。A亚基和c-环之间的大空腔为质子创造了一个细胞质半通道。C-环上携带质子的Glu残基分布不对称，c‘’亚基的Glu残基与a亚基的Arg735相互作用。这种结构表明c-环的质子化和去质子化，由ATP水解酶驱动的旋转导致细胞质半通道处的Glu残基质子化，随后管腔半通道处的Glu残基去质子化。
Vacuolar-type ATPases (V-ATPases) are ATP-powered proton pumps involved in processes such as endocytosis, lysosomal degradation, secondary transport, TOR signalling, and osteoclast and kidney function. ATP hydrolysis in the soluble catalytic V-1 region drives proton translocation through the membrane-embedded V-O region via rotation of a rotor subcomplex. Variability in the structure of the intact enzyme has prevented construction of an atomic model for the membrane-embedded motor of any rotary ATPase(1-5). We induced dissociation and auto-inhibition of the V1 and VO regions of the V-ATPase by starving the yeast Saccharomyces cerevisiae(6,7), allowing us to obtain a resolution electron cryomicroscopy map of the VO complex and build atomic models for the majority of its subunits. The analysis reveals the structures of subunits ac(8)c'c '' de and a protein that we identify and propose to be a new subunit (subunit f). A large cavity between subunit a and the c-ring creates a cytoplasmic half-channel for protons. The c-ring has an asymmetric distribution of proton-carrying Glu residues, with the Glu residue of subunit c '' interacting with Arg735 of subunit a. The structure suggests sequential protonation and deprotonation of the c-ring, with ATP-hydrolysis-driven rotation causing protonation of a Glu residue at the cytoplasmic half-channel and subsequent deprotonation of a Glu residue at a luminal half-channel.