Kawasaki-Nishi S, Nishi T, Forgac M
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
J Biol Chem. 2001 May 25;276(21):17941-8. doi: 10.1074/jbc.M010790200. Epub 2001 Mar 2.
The 100 kDa a-subunit of the yeast vacuolar (H(+))-ATPase (V-ATPase) is encoded by two genes, VPH1 and STV1. These genes encode unique isoforms of the a-subunit that have previously been shown to reside in different intracellular compartments in yeast. Vph1p localizes to the central vacuole, whereas Stv1p is present in some other compartment, possibly the Golgi or endosomes. To compare the properties of V-ATPases containing Vph1p or Stv1p, Stv1p was expressed at higher than normal levels in a strain disrupted in both genes, under which conditions V-ATPase complexes containing Stv1p appear in the vacuole. Complexes containing Stv1p showed lower assembly with the peripheral V(1) domain than did complexes containing Vph1p. When corrected for this lower degree of assembly, however, V-ATPase complexes containing Vph1p and Stv1p had similar kinetic properties. Both exhibited a K(m) for ATP of about 250 microm, and both showed resistance to sodium azide and vanadate and sensitivity to nanomolar concentrations of concanamycin A. Stv1p-containing complexes, however, showed a 4-5-fold lower ratio of proton transport to ATP hydrolysis than Vph1p-containing complexes. We also compared the ability of V-ATPase complexes containing Vph1p or Stv1p to undergo in vivo dissociation in response to glucose depletion. Vph1p-containing complexes present in the vacuole showed dissociation in response to glucose depletion, whereas Stv1p-containing complexes present in their normal intracellular location (Golgi/endosomes) did not. Upon overexpression of Stv1p, Stv1p-containing complexes present in the vacuole showed glucose-dependent dissociation. Blocking delivery of Vph1p-containing complexes to the vacuole in vps21Delta and vps27Delta strains caused partial inhibition of glucose-dependent dissociation. These results suggest that dissociation of the V-ATPase complex in vivo is controlled both by the cellular environment and by the 100-kDa a-subunit isoform present in the complex.
酵母液泡（H⁺）-ATP酶（V-ATP酶）的100 kDa α亚基由两个基因VPH1和STV1编码。这些基因编码α亚基的独特异构体，先前已证明它们存在于酵母的不同细胞内区室中。Vph1p定位于中央液泡，而Stv1p存在于其他一些区室中，可能是高尔基体或内体。为了比较含有Vph1p或Stv1p的V-ATP酶的特性，在两个基因均被破坏的菌株中以高于正常水平表达Stv1p，在这种条件下，含有Stv1p的V-ATP酶复合物出现在液泡中。与含有Vph1p的复合物相比，含有Stv1p的复合物与外周V1结构域的组装程度较低。然而，当校正这种较低的组装程度时，含有Vph1p和Stv1p的V-ATP酶复合物具有相似的动力学特性。两者对ATP的Kₘ约为250 μM，均表现出对叠氮化钠和钒酸盐的抗性以及对纳摩尔浓度的 concanamycin A的敏感性。然而，含有Stv1p的复合物的质子转运与ATP水解的比率比含有Vph1p的复合物低4-5倍。我们还比较了含有Vph1p或Stv1p的V-ATP酶复合物在体内响应葡萄糖耗尽而发生解离的能力。液泡中存在的含有Vph1p的复合物在响应葡萄糖耗尽时表现出解离，而存在于其正常细胞内位置（高尔基体/内体）的含有Stv1p的复合物则没有。在Stv1p过表达时，液泡中存在的含有Stv1p的复合物表现出葡萄糖依赖性解离。在vps21Δ和vps27Δ菌株中阻断含有Vph1p的复合物向液泡的转运导致葡萄糖依赖性解离的部分抑制。这些结果表明，V-ATP酶复合物在体内的解离受细胞环境和复合物中存在的100 kDa α亚基异构体的控制。
