Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210.
Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210
Genetics. 2018 Feb;208(2):655-672. doi: 10.1534/genetics.117.300594. Epub 2017 Dec 18.
Loss of V-ATPase activity in organelles, whether through V-ATPase inhibition or V-ATPase () mutations, triggers a compensatory downregulation of the essential plasma membrane proton pump Pma1 in We have previously determined that the α-arrestin Rim8 and ubiquitin ligase Rsp5 are essential for Pma1 ubiquination and endocytosis in response to loss of V-ATPase activity. Here, we show that Pma1 endocytosis in V-ATPase mutants does not require Rim101 pathway components upstream and downstream of Rim8, indicating that Rim8 is acting independently in Pma1 internalization. We find that two phosphatases, the calcium-responsive phosphatase calcineurin and the glucose-sensitive phosphatase Glc7 (PP1), and one of the Glc7 regulatory subunits Reg1, exhibit negative synthetic genetic interactions with mutants, and demonstrate that both phosphatases are essential for ubiquitination and endocytic downregulation of Pma1 in these mutants. Although both acute and chronic loss of V-ATPase activity trigger the internalization of ∼50% of surface Pma1, a comparable reduction in Pma1 expression in a mutant neither compensates for loss of V-ATPase activity nor stops further Pma1 endocytosis. The results indicate that the cell surface level of Pma1 is not directly sensed and that internalized Pma1 may play a role in compensating for loss of V-ATPase-dependent acidification. Taken together, these results provide new insights into cross talk between two major proton pumps central to cellular pH control.
细胞器中 V-ATPase 活性的丧失，无论是通过 V-ATPase 抑制还是 V-ATPase () 突变，都会触发必需的质膜质子泵 Pma1 在中的代偿性下调。我们之前已经确定，α-抑制素 Rim8 和泛素连接酶 Rsp5 对于 Pma1 的泛素化和内吞作用是必需的，以响应 V-ATPase 活性的丧失。在这里，我们表明，V-ATPase 突变体中 Pma1 的内吞作用不需要 Rim8 上下游的 Rim101 途径成分，这表明 Rim8 独立于 Pma1 的内化作用。我们发现两种磷酸酶，钙反应性磷酸酶钙调神经磷酸酶和葡萄糖敏感磷酸酶 Glc7（PP1），以及 Glc7 的一个调节亚基 Reg1，与 突变体表现出负的合成遗传相互作用，并证明这两种磷酸酶对于这些突变体中 Pma1 的泛素化和内吞下调都是必需的。虽然急性和慢性 V-ATPase 活性丧失都会触发约 50%的表面 Pma1 的内化，但在 突变体中 Pma1 表达的类似减少既不能补偿 V-ATPase 活性的丧失，也不能阻止进一步的 Pma1 内吞作用。结果表明，Pma1 的细胞表面水平不是直接感知的，并且内化的 Pma1 可能在补偿 V-ATPase 依赖性酸化的丧失方面发挥作用。综上所述，这些结果为两个对细胞 pH 控制至关重要的主要质子泵之间的交叉对话提供了新的见解。
