Liu Q, Feng Y, Forgac M
Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
J Biol Chem. 1994 Dec 16;269(50):31592-7.
We have previously shown that the 50-kDa subunit of the clathrin assembly complex AP-2 (AP50) stoichiometrically binds to and is immunoprecipitated with the vacuolar (H+)-ATPase (V-ATPase) from clathrin-coated vesicles (Myers, M., and Forgac, M. (1993) J. Biol. Chem. 268, 9184-9186). We now report that treatment of stripped coated vesicles with cystine results in a purified V-ATPase complex lacking the AP50 polypeptide. Removal of AP50 can be reversed upon treatment of the vesicles with dithiothreitol. Removal of AP50 reduces the ATPase activity of the purified V-ATPase by 90% relative to the enzyme containing AP50. This inhibition is not reversed upon treatment of the AP50-depleted enzyme with dithiothreitol in the absence of AP50. The reconstituted V-ATPase depleted of AP50 is devoid of ATP-dependent proton transport activity. We observe further that the peripheral V1 subunits are unable to reassemble onto the integral V0 domain in the absence of AP50. The addition of purified AP-2 containing the AP50 polypeptide restores the ability of the V1 subunits to assemble with the V0 sector to give a V-ATPase complex that is functional in ATP-dependent proton transport. These results indicate that the AP50 polypeptide is necessary for both activity and in vitro reassembly of the V-ATPase complex.
我们之前已经表明，网格蛋白组装复合体AP-2（AP50）的50 kDa亚基以化学计量方式与网格蛋白包被小泡中的液泡（H⁺）-ATP酶（V-ATP酶）结合并被共免疫沉淀（迈尔斯，M.，和福加克，M.（1993年）《生物化学杂志》268，9184 - 9186）。我们现在报告，用胱氨酸处理去除蛋白的包被小泡会产生一种缺乏AP50多肽的纯化V-ATP酶复合体。用二硫苏糖醇处理小泡后，AP50的去除可以逆转。去除AP50后，纯化的V-ATP酶的ATP酶活性相对于含有AP50的酶降低了90%。在没有AP50的情况下，用二硫苏糖醇处理去除AP50的酶后，这种抑制作用不会逆转。去除AP50的重组V-ATP酶缺乏ATP依赖的质子转运活性。我们进一步观察到，在没有AP50的情况下，外周V1亚基无法重新组装到完整的V0结构域上。添加含有AP50多肽的纯化AP-2可恢复V1亚基与V0区段组装形成在ATP依赖的质子转运中起作用的V-ATP酶复合体的能力。这些结果表明，AP50多肽对于V-ATP酶复合体的活性和体外重新组装都是必需的。
