Qi Jie, Forgac Michael
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
J Biol Chem. 2007 Aug 24;282(34):24743-51. doi: 10.1074/jbc.M700663200. Epub 2007 Jun 12.
One mechanism of regulating V-ATPase activity in vivo involves reversible dissociation into its component V(1) and V(0) domains, which in yeast occurs in response to glucose depletion. V-ATPase complexes containing the Vph1p isoform of subunit a (VCC) are targeted to the vacuole, and Stv1p-containing complexes (SCC) are targeted to the Golgi. Overexpression of Stv1p results in mistargeting of SCC to the vacuole. We have investigated the role of the a subunit isoform and cellular environment in controlling dissociation using vacuolar protein sorting (vps) mutants that accumulate proteins in either the prevacuolar compartment (PVC) (vps27Delta) or a post-Golgi compartment (PGC) (vps21Delta). Dissociation of both VCC and SCC depends upon cellular environment, with dissociation most complete in the vacuole and least complete in the PVC. The dependence of dissociation on V-ATPase activity was also investigated using both concanamycin and inactivating mutations. Concanamycin partly blocks dissociation of both VCC and SCC in all three compartments, with inhibition generally greater for SCC than VCC. The R735Q mutant of Vph1p results in loss of both ATPase and proton transport, whereas the R735K mutant lacks proton transport but has 10% of wild type ATPase activity. For VCC in the vacuole, dissociation is completely blocked for the R735Q but not the R735K mutant. Significant dissociation of VCC is observed for both mutants in the PVC and PGC, indicating that V-ATPase activity is not absolutely required for dissociation. Similar results were obtained for SCC, although dissociation of SCC is again generally more sensitive to activity than VCC. These results suggest that the cellular environment is important both in controlling in vivo dissociation of the V-ATPase and the dependence of this process on catalytic activity. Moreover, catalytic activity is not absolutely required for V-ATPase dissociation.
体内调节V-ATP酶活性的一种机制涉及可逆解离成其组成部分V(1)和V(0)结构域，这在酵母中是对葡萄糖耗尽的反应。含有亚基a的Vph1p同种型的V-ATP酶复合物（VCC）定位于液泡，而含有Stv1p的复合物（SCC）定位于高尔基体。Stv1p的过表达导致SCC错定位于液泡。我们使用液泡蛋白分选（vps）突变体研究了a亚基同种型和细胞环境在控制解离中的作用，这些突变体在液泡前区室（PVC）（vps27Delta）或高尔基体后区室（PGC）（vps21Delta）中积累蛋白质。VCC和SCC的解离均取决于细胞环境，在液泡中解离最完全，在PVC中最不完全。还使用了 concanamycin 和失活突变研究了解离对V-ATP酶活性的依赖性。Concanamycin 部分阻断了所有三个区室中VCC和SCC的解离，对SCC的抑制通常比对VCC更大。Vph1p的R735Q突变体导致ATP酶和质子转运丧失，而R735K突变体缺乏质子转运但具有野生型ATP酶活性的10%。对于液泡中的VCC，R735Q突变体完全阻断了解离，但R735K突变体没有。在PVC和PGC中，两个突变体均观察到VCC的显著解离，表明V-ATP酶活性不是解离所绝对必需的。对于SCC也获得了类似的结果，尽管SCC的解离通常再次比VCC对活性更敏感。这些结果表明，细胞环境在控制V-ATP酶的体内解离以及该过程对催化活性的依赖性方面都很重要。此外，V-ATP酶解离并非绝对需要催化活性。
