Identification of novel cellular partners involved in controlling reversible dissociation of V-ATPases in vivo

真核细胞已经进化出一个ATP依赖性质子泵家族，称为液泡H+-ATP酶（V-ATP酶）。V-ATP酶存在于多种细胞内区室中，其中它们通过以ATP为代价将质子泵送穿过膜来负责pH稳态。细胞器的酸化在膜运输、蛋白质降解、小分子的偶联运输和各种病原体的进入中是必不可少的。此外，V-ATP酶在正常生理过程以及许多人类疾病中起着至关重要的作用。V-ATP酶活性的快速有效调节机制涉及V1和VO两个功能结构域的解离和缔合。迄今为止，这一过程在酵母中得到了最好的表征，其中V-ATP酶响应于葡萄糖消耗而可逆地分解。虽然Ras/cAMP/PKA（蛋白激酶A）通路最近已被证明在酵母中的V-ATP酶的可逆解离的调节中起作用，但是对于将PKA连接到该过程的蛋白质一无所知。也不知道V-ATP酶或相关蛋白质的亚基是否是PKA的底物。该建议将寻找新的蛋白下游PKA参与可逆解离使用一种新的遗传筛选，确定突变体在这一过程中有缺陷。还将进行实验以测试与复合物相关的V-ATP酶亚基或蛋白质在可逆解离期间是否被磷酸化或去磷酸化。

Eukaryotic cells have evolved a family of ATP-dependent proton pumps known as the vacuolar H+-ATPases (V-ATPases). V-ATPases are present in a variety of intracellular compartments where they are responsible for pH homeostasis by pumping protons across the membrane at the expense of ATP. Acidification of organelles is essential in membrane trafficking, protein degradation, coupled transport of small molecules, and the entry of various pathogens. Furthermore, V-ATPases play a crucial role for normal physiological processes as well as in a number of human diseases. A rapid and effective mechanism for regulating the activity of V-ATPases involves dissociation and association of the two functional domains, V1 and VO. To date, this process is best characterized in yeast, where V-ATPases are reversibly disassembled in response to glucose depletion. Although the Ras/cAMP/PKA (protein kinase A) pathway has recently been shown to function in regulation of reversible dissociation of the V-ATPase in yeast, nothing is known about the proteins that connect PKA to this process. It is also not known whether subunits of the V-ATPase or associated proteins are substrates of PKA. This proposal will search for novel proteins downstream of PKA that are involved in reversible dissociation using a novel genetic screen that identifies mutants defective in this process. Experiments will also be performed to test whether V-ATPase subunits or proteins associated with the complex become phosphorylated or dephosphorylated during reversible dissociation.