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类依赖于三磷酸腺苷的质子泵，称为液泡H+-ATPase(V-ATPase)。V-ATPase存在于细胞内的各种隔室中，它们通过泵送质子穿过细胞膜而损害ATP，从而负责pH的动态平衡。细胞器的酸化在膜运输、蛋白质降解、小分子的耦合运输和各种病原体的进入过程中是必不可少的。此外，V-ATPase在正常的生理过程中以及在许多人类疾病中都发挥着至关重要的作用。一种快速而有效的调节V-ATPase活性的机制涉及V1和VO两个功能结构域的解离和关联。到目前为止，这一过程在酵母中表现得最好，在酵母中，V-ATPase在葡萄糖耗尽时被可逆分解。虽然Ras/cAMP/PKA(蛋白激酶A)通路最近被证明在调节酵母中V-ATPase的可逆解离中起作用，但对于将PKA连接到这一过程的蛋白质却知之甚少。也不知道V-ATPase的亚基或相关蛋白是否是PKA的底物。这项提议将使用一种新的基因筛选来寻找参与可逆解离的PKA下游的新蛋白，以识别在这一过程中存在缺陷的突变体。还将进行实验，以测试与该复合体相关的V-ATPase亚单位或蛋白质在可逆解离过程中是否发生磷酸化或去磷酸化。

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.