Liu Q, Leng X H, Newman P R, Vasilyeva E, Kane P M, Forgac M
Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
J Biol Chem. 1997 May 2;272(18):11750-6. doi: 10.1074/jbc.272.18.11750.
To investigate the function of residues at the catalytic nucleotide binding site of the V-ATPase, we have carried out site-directed mutagenesis of the VMA1 gene encoding the A subunit of the V-ATPase in yeast. Of the three cysteine residues that are conserved in all A subunits sequenced thus far, two (Cys284 and Cys539) appear essential for correct folding or stability of the A subunit. Mutation of the third cysteine (Cys261), located in the glycine-rich loop, to valine, generated an enzyme that was fully active but resistant to inhibition by N-ethylmalemide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and oxidation. To test the role of disulfide bond formation in regulation of vacuolar acidification in vivo, we have also determined the effect of the C261V mutant on targeting and processing of the soluble vacuolar protein carboxypeptidase Y. No difference in carboxypeptidase Y targeting or processing is observed between the wild type and C261V mutant, suggesting that disulfide bond formation in the V-ATPase A subunit is not essential for controlling vacuolar acidification in the Golgi. In addition, fluid phase endocytosis of Lucifer Yellow, quinacrine staining of acidic intracellular compartments and cell growth are indistinguishable in the C261V and wild type cells. Mutation of G250D in the glycine-rich loop also resulted in destabilization of the A subunit, whereas mutation of the lysine residue in this region (K263Q) gave a V-ATPase complex which showed normal levels of A subunit on the vacuolar membrane but was unstable to detergent solubilization and isolation and was totally lacking in V-ATPase activity. By contrast, mutation of the acidic residue, which has been postulated to play a direct catalytic role in the homologous F-ATPases (E286Q), had no effect on stability or assembly of the V-ATPase complex, but also led to complete loss of V-ATPase activity. The E286Q mutant showed labeling by 2-azido-[32P]ATP that was approximately 60% of that observed for wild type, suggesting that mutation of this glutamic acid residue affected primarily ATP hydrolysis rather than nucleotide binding.
为了研究V-ATP酶催化核苷酸结合位点残基的功能，我们对酵母中编码V-ATP酶A亚基的VMA1基因进行了定点诱变。在迄今为止测序的所有A亚基中保守的三个半胱氨酸残基中，有两个（Cys284和Cys539）对于A亚基的正确折叠或稳定性似乎至关重要。位于富含甘氨酸环中的第三个半胱氨酸（Cys261）突变为缬氨酸，产生了一种完全有活性但对N-乙基马来酰胺、7-氯-4-硝基苯-2-恶唑-1,3-二唑和氧化具有抗性的酶。为了测试二硫键形成在体内液泡酸化调节中的作用，我们还确定了C261V突变体对可溶性液泡蛋白羧肽酶Y的靶向和加工的影响。在野生型和C261V突变体之间未观察到羧肽酶Y靶向或加工的差异，这表明V-ATP酶A亚基中的二硫键形成对于控制高尔基体中的液泡酸化不是必需的。此外，在C261V和野生型细胞中，荧光素黄的液相内吞作用、酸性细胞内区室的喹吖因染色和细胞生长没有区别。富含甘氨酸环中G250D的突变也导致A亚基不稳定，而该区域赖氨酸残基的突变（K263Q）产生了一种V-ATP酶复合物，其在液泡膜上显示出正常水平的A亚基，但对去污剂溶解和分离不稳定且完全缺乏V-ATP酶活性。相比之下，据推测在同源F-ATP酶中起直接催化作用的酸性残基的突变（E286Q）对V-ATP酶复合物的稳定性或组装没有影响，但也导致V-ATP酶活性完全丧失。E286Q突变体显示2-叠氮基-[32P]ATP的标记约为野生型观察值的60%，这表明该谷氨酸残基的突变主要影响ATP水解而不是核苷酸结合。
