Xie X S
Division of Molecular Transport, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9121, USA.
J Biol Chem. 1996 Nov 29;271(48):30980-5.
The vacuolar-type proton pump of clathrin-coated vesicles is composed of two general domains, a peripheral, catalytic sector (VC) and a transmembranous proton channel (VB). In its native form, the enzyme can hydrolyze both MgATP and CaATP, whereas VC, when separated from VB, loses its MgATPase activity and switches to a state that can hydrolyze only CaATP. Further dissociation of VC results in subcomplexes that are depleted of one or more subunits and lack ATPase activity altogether. Reconstitution of recombinant subunits to these biochemically prepared subcomplexes has demonstrated the necessity of polypeptides of 70, 58, 40, and 33 kDa (subunits A, B, C, and E, respectively) for CaATPase activity of the VC complex. The current studies demonstrate that mixtures of these four recombinant subunits cannot support CaATPase activity in the absence of a biochemically prepared subcomplex. Investigation of the other components required for ATPase activity has led to the identification of three additional polypeptides present in preparations of VC, with apparent molecular masses of 15, 14, and 10 kDa. Each of these proteins was found to activate ATPase activity of mixtures of subunits A, B, C, and E. In addition, ATPase reconstituted from these individual subunits hydrolyses ATP, not only in the presence of Ca2+ but also in the presence of Mg2+. Investigation of the individual properties of these three subunits revealed that the 10-kDa polypeptide is subunit F, as determined by immunoblot analysis. This subunit had no effect on MgATPase activity of VC but stimulated CaATPase activity 6-fold in the presence of subunit D. Under optimal conditions the 14-kDa component resulted in a 10-fold stimulation and the 15-kDa component a 20-fold stimulation of MgATPase activity; based on this observation, the 14- and 15-kDa polypeptides were named subunits G and H, respectively. In addition, proton pumping activity was reconstituted through the reassembly of subunits A-H with VB and SFD, a previously described pump component composed of polypeptides of 50 and 57 kDa (Xie, X.-S, Crider, B.P., Ma, Y. M., and Stone, D. K. (1994) J. Biol. Chem. 269, 25809-25815). Together, these experiments completely define the catalytic center of the vacuolar proton pump of clathrin-coated vesicles.
网格蛋白包被小泡的液泡型质子泵由两个一般结构域组成，一个外周催化区（VC）和一个跨膜质子通道（VB）。在其天然形式下，该酶既能水解MgATP也能水解CaATP，而当VC与VB分离时，它会失去MgATP酶活性并转变为只能水解CaATP的状态。VC的进一步解离会产生亚复合物，这些亚复合物缺少一个或多个亚基并且完全缺乏ATP酶活性。将重组亚基重新组装到这些通过生化方法制备的亚复合物上，已证明70、58、40和33 kDa的多肽（分别为亚基A、B、C和E）对于VC复合物的CaATP酶活性是必需的。目前的研究表明，在没有通过生化方法制备的亚复合物的情况下，这四种重组亚基的混合物不能支持CaATP酶活性。对ATP酶活性所需的其他成分的研究导致在VC制剂中鉴定出另外三种多肽，其表观分子量分别为15、14和10 kDa。发现这些蛋白质中的每一种都能激活亚基A、B、C和E混合物的ATP酶活性。此外，由这些单个亚基重构的ATP酶不仅在Ca2+存在时，而且在Mg2+存在时都能水解ATP。对这三个亚基各自特性的研究表明，通过免疫印迹分析确定10 kDa的多肽是亚基F。该亚基对VC 的MgATP酶活性没有影响，但在亚基D存在时能将CaATP酶活性刺激6倍。在最佳条件下，14 kDa的成分使MgATP酶活性提高10倍，15 kDa的成分使MgATP酶活性提高20倍；基于这一观察结果，14 kDa和15 kDa的多肽分别被命名为亚基G和H。此外，通过将亚基A-H与VB和SFD重新组装来重构质子泵活性，SFD是一种先前描述的由50和57 kDa多肽组成的泵成分（谢，X.-S，克里德，B.P.，马，Y.M.和斯通，D.K.（1994年）《生物化学杂志》269，25809-25815）。总之，这些实验完全确定了网格蛋白包被小泡的液泡质子泵的催化中心。
