Yeast vacuoles are acidified by the V-ATPase, a protein complex comprised of the membrane embedded VO complex and the soluble cytoplasmic V1 complex. The assembly of the V1-VO holoenzyme is required for the transfer of H+ into the vacuole lumen for acidification. The assembly of the V1-VO holoenzyme is stabilized by the lipid phosphatidylinositol 3,5-bisphospate (PI(3,5)P2) made by the PI3P 5-kinase Fab1/PIKfyve. The absence of PI(3,5)P2 leads to the dissociation of the V1 complex from the membrane. Separately, PI(3,5)P2 has been shown to modulate Ca2+ transport across the vacuole membrane during fission and fusion. Here we examined whether the regulation of H+ and Ca2+ by PI(3,5)P2 are interdependent. We show that modulating extraluminal Ca2+ concentrations inhibit V-ATPase activity. As extraluminal CaCl2 levels are raised, the activity of H+ pumping is reduced. Conversely, chelating free Ca2+ with EGTA stimulated vacuole acidification. Not only did Ca2+ levels affect H+ translocation, we also show that blocking V-ATPase activity inhibited Ca2+ transport into the vacuole lumen. Together, these data illustrate that Ca2+ transport and V-ATPase regulation are interconnected through the modulation of vacuolar lipid profiles.

中文翻译：

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液泡钙转运和V-ATPase活性的相互调节，以及磷脂酰肌醇3,5-双磷酸酯的影响

酵母液泡被V-ATPase酸化，V-ATPase是一种蛋白质复合物，由膜嵌入的VO复合物和可溶性细胞质V1复合物组成。为了将H +转移到液泡腔中进行酸化，需要V1-VO全酶的组装。通过由PI3P 5激酶Fab1 / PIKfyve制造的脂质磷脂酰肌醇3,5-二磷酸酯（PI（3,5）P2）可以稳定V1-VO全酶的组装。PI（3,5）P2的缺乏导致V1复合物从膜上解离。另外，PI（3,5）P2已显示在裂变和融合过程中调节Ca2 +跨液泡膜的运输。在这里，我们检查了PI（3,5）P2对H +和Ca2 +的调节是否相互依赖。我们表明调节腔外Ca 2 +浓度抑制V-ATPase活性。随着腔外CaCl2含量的升高，H +抽运的活动减少了。相反，用EGTA螯合游离Ca2 +可刺激液泡酸化。Ca2 +水平不仅影响H +易位，而且还显示阻断V-ATPase活性抑制Ca2 +转运入液泡腔。总之，这些数据说明Ca2 +转运和V-ATPase调节通过液泡脂质分布的调节相互联系。