Subunit rotation of vacuolar-type proton pumping ATPase -: Relative rotation of the G and c subunits
Subunit rotation of vacuolar-type proton pumping ATPase -: Relative rotation of the G and c subunits
复制标题
DOI：
10.1074/jbc.m302756200
复制
发表时间：
2003-06-27
影响因子：
4.8
通讯作者：
Futai, M
中科院分区：
文献类型：
--
作者：
Hirata, T;Iwamoto-Kihara, A;Futai, M
空泡型ATPase V1V0(V-ATPase)广泛存在于真核细胞的内膜细胞器中。在本研究中，我们在酿酒酵母V-ATPase的c和G亚基上分别引入了一个His标签和一个生物素标签。利用这种工程酶，我们直接观察到当酶通过c亚基固定在玻璃表面时，附着在G亚基上的肌动蛋白细丝连续逆时针旋转。V-ATPase产生的扭矩基本上与F-ATPase(ATP合成酶)相同。刀豆素和硝酸盐对旋转有抑制作用，叠氮无抑制作用。这些结果表明，V-ATPase和F-ATPase具有共同的旋转催化作用。
Vacuolar-type ATPases V1V0 (V-ATPases) are found ubiquitously in the endomembrane organelles of eukaryotic cells. In this study, we genetically introduced a His tag and a biotin tag onto the c and G subunits, respectively, of Saccharomyces cerevisiae V-ATPase. Using this engineered enzyme, we observed directly the continuous counter-clockwise rotation of an actin filament attached to the G subunit when the enzyme was immobilized on a glass surface through the c subunit. V-ATPase generated essentially the same torque as the F-ATPase (ATP synthase). The rotation was inhibited by concanamycin and nitrate but not by azide. These results demonstrated that the V- and F-ATPase carry out a common rotational catalysis.