Zhang Zhenyu, Zheng Yesha, Mazon Hortense, Milgrom Elena, Kitagawa Norton, Kish-Trier Erik, Heck Albert J R, Kane Patricia M, Wilkens Stephan
Department of Biochemistry, University of California, Riverside, California 92521, USA.
J Biol Chem. 2008 Dec 19;283(51):35983-95. doi: 10.1074/jbc.M805345200. Epub 2008 Oct 27.
The subunit architecture of the yeast vacuolar ATPase (V-ATPase) was analyzed by single particle transmission electron microscopy and electrospray ionization (ESI) tandem mass spectrometry. A three-dimensional model of the intact V-ATPase was calculated from two-dimensional projections of the complex at a resolution of 25 angstroms. Images of yeast V-ATPase decorated with monoclonal antibodies against subunits A, E, and G position subunit A within the pseudo-hexagonal arrangement in the V1, the N terminus of subunit G in the V1-V0 interface, and the C terminus of subunit E at the top of the V1 domain. ESI tandem mass spectrometry of yeast V1-ATPase showed that subunits E and G are most easily lost in collision-induced dissociation, consistent with a peripheral location of the subunits. An atomic model of the yeast V-ATPase was generated by fitting of the available x-ray crystal structures into the electron microscopy-derived electron density map. The resulting atomic model of the yeast vacuolar ATPase serves as a framework to help understand the role the peripheral stalk subunits are playing in the regulation of the ATP hydrolysis driven proton pumping activity of the vacuolar ATPase.
通过单颗粒透射电子显微镜和电喷雾电离（ESI）串联质谱分析了酵母液泡ATP酶（V-ATP酶）的亚基结构。从复合物的二维投影以25埃的分辨率计算出完整V-ATP酶的三维模型。用针对亚基A、E和G的单克隆抗体修饰的酵母V-ATP酶图像，将亚基A定位在V1的假六边形排列内，将亚基G的N端定位在V1-V0界面，将亚基E的C端定位在V1结构域顶部。酵母V1-ATP酶的ESI串联质谱显示，亚基E和G在碰撞诱导解离中最容易丢失，这与这些亚基的外周位置一致。通过将可用的X射线晶体结构拟合到电子显微镜衍生的电子密度图中，生成了酵母V-ATP酶的原子模型。所得的酵母液泡ATP酶原子模型作为一个框架，有助于理解外周柄亚基在调节液泡ATP酶的ATP水解驱动质子泵活性中所起的作用。
