Couoh-Cardel Sergio, Milgrom Elena, Wilkens Stephan
From the Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210.
From the Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210
J Biol Chem. 2015 Nov 13;290(46):27959-71. doi: 10.1074/jbc.M115.662494. Epub 2015 Sep 28.
The membrane sector (Vo) of the proton pumping vacuolar ATPase (V-ATPase, V1Vo-ATPase) from Saccharomyces cerevisiae was purified to homogeneity, and its structure was characterized by EM of single molecules and two-dimensional crystals. Projection images of negatively stained Vo two-dimensional crystals showed a ring-like structure with a large asymmetric mass at the periphery of the ring. A cryo-EM reconstruction of Vo from single-particle images showed subunits a and d in close contact on the cytoplasmic side of the proton channel. A comparison of three-dimensional reconstructions of free Vo and Vo as part of holo V1Vo revealed that the cytoplasmic N-terminal domain of subunit a (aNT) must undergo a large conformational change upon enzyme disassembly or (re)assembly from Vo, V1, and subunit C. Isothermal titration calorimetry using recombinant subunit d and aNT revealed that the two proteins bind each other with a Kd of ~5 μm. Treatment of the purified Vo sector with 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] resulted in selective release of subunit d, allowing purification of a VoΔd complex. Passive proton translocation assays revealed that both Vo and VoΔd are impermeable to protons. We speculate that the structural change in subunit a upon release of V1 from Vo during reversible enzyme dissociation plays a role in blocking passive proton translocation across free Vo and that the interaction between aNT and d seen in free Vo functions to stabilize the Vo sector for efficient reassembly of V1Vo.
酿酒酵母质子泵液泡ATP酶（V-ATP酶，V1Vo-ATP酶）的膜区（Vo）被纯化至同质，并通过单分子和二维晶体的电子显微镜对其结构进行了表征。负染的Vo二维晶体的投影图像显示出一种环状结构，在环的周边有一个大的不对称质量。从单颗粒图像对Vo进行的冷冻电镜重建显示，亚基a和d在质子通道的细胞质侧紧密接触。对游离Vo和作为全酶V1Vo一部分的Vo的三维重建进行比较，结果表明，在酶从Vo、V1和亚基C进行拆卸或（重新）组装时，亚基a的细胞质N端结构域（aNT）必须经历大的构象变化。使用重组亚基d和aNT进行等温滴定量热法研究表明，这两种蛋白质以~5μm的解离常数相互结合。用1-棕榈酰-2-羟基-sn-甘油-3-[磷酸-rac-（1-甘油）]处理纯化的Vo区，导致亚基d选择性释放，从而能够纯化VoΔd复合物。被动质子转运试验表明，Vo和VoΔd对质子均不可渗透。我们推测，在可逆酶解离过程中V1从Vo释放时亚基a的结构变化在阻止质子通过游离Vo的被动转运中起作用，并且在游离Vo中观察到的aNT与d之间的相互作用起到稳定Vo区的作用，以实现V1Vo的高效重新组装。
