Vacuolar and plasma membrane proton pumps collaborate to achieve cytosolic pH homeostasis in yeast
Vacuolar and plasma membrane proton pumps collaborate to achieve cytosolic pH homeostasis in yeast
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DOI：
10.1074/jbc.m710470200
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发表时间：
2008-07-18
影响因子：
4.8
通讯作者：
Kane, Patricia
中科院分区：
文献类型：
--
作者：
Martinez-Munoz, Gloria A.;Kane, Patricia
极性质子转运ATP酶（V-ATP酶）在真核细胞的细胞器酸化中起着重要作用。为了解决的作用，酵母V-ATP酶在液泡和胞质pH值的稳态，比例pH值敏感的荧光团的液泡或胞质被引入到野生型细胞和VMA突变体，缺乏V-ATP酶亚基。短暂的葡萄糖剥夺的野生型细胞响应葡萄糖除了空泡酸化和细胞质碱化，和随后加入的K+离子增加的pH值的空泡和细胞质。相比之下，葡萄糖的加入导致vma突变体和野生型细胞用V-ATP酶抑制剂康卡霉素A处理后液泡pH值升高。在vma突变体中，细胞溶质pH稳态也被显著扰乱。即使在细胞外的pH值为5，其生长的最佳条件，胞质pH值低得多，葡萄糖的响应是较小的突变体。在来自vma突变体的质膜组分中，质膜质子泵Pma 1 p的活性比来自野生型细胞的组分低65-75%。免疫荧光显微镜证实质膜Pma 1 p水平下降，增加Pma 1 p在液泡和其他隔室的突变体。Pma 1 p没有错误定位在康卡霉素处理的细胞，但在所有条件下，细胞溶质pH值的显着降低仍然观察到。我们建议，短期，V-ATP酶活性是必不可少的液泡酸化响应葡萄糖代谢和有效的胞质pH值的稳态，长期，V-ATP酶是重要的Pma 1 p在质膜上的稳定定位。
Vacuolar proton-translocating ATPases (V-ATPases) play a central role in organelle acidification in all eukaryotic cells. To address the role of the yeast V-ATPase in vacuolar and cytosolic pH homeostasis, ratiometric pH-sensitive fluorophores specific for the vacuole or cytosol were introduced into wild-type cells and vma mutants, which lack V-ATPase subunits. Transiently glucose-deprived wild-type cells respond to glucose addition with vacuolar acidification and cytosolic alkalinization, and subsequent addition of K+ ion increases the pH of both the vacuole and cytosol. In contrast, glucose addition results in an increase in vacuolar pH in both vma mutants and wild-type cells treated with the V-ATPase inhibitor concanamycin A. Cytosolic pH homeostasis is also significantly perturbed in the vma mutants. Even at extracellular pH5, conditions optimal for their growth, cytosolic pH was much lower, and response to glucose was smaller in the mutants. In plasma membrane fractions from the vma mutants, activity of the plasma membrane proton pump, Pma1p, was 65-75% lower than in fractions from wildtype cells. Immunofluorescence microscopy confirmed decreased levels of plasma membrane Pma1p and increased Pma1p at the vacuole and other compartments in the mutants. Pma1p was not mislocalized in concanamycin-treated cells, but a significant reduction in cytosolic pH under all conditions was still observed. We propose that short-term, V-ATPase activity is essential for both vacuolar acidification in response to glucose metabolism and for efficient cytosolic pH homeostasis, and long-term, V-ATPases are important for stable localization of Pma1p at the plasma membrane.