Department of Biochemistry, University of Lausanne, Ch. des Boveresses 155, 1066 Epalinges, Switzerland.
Sci Rep. 2016 Jul 1;6:29045. doi: 10.1038/srep29045.
The V-ATPase is a proton pump consisting of a membrane-integral V0 sector and a peripheral V1 sector, which carries the ATPase activity. In vitro studies of yeast vacuole fusion and evidence from worms, flies, zebrafish and mice suggested that V0 interacts with the SNARE machinery for membrane fusion, that it promotes the induction of hemifusion and that this activity requires physical presence of V0 rather than its proton pump activity. A recent in vivo study in yeast has challenged these interpretations, concluding that fusion required solely lumenal acidification but not the V0 sector itself. Here, we identify the reasons for this discrepancy and reconcile it. We find that acute pharmacological or physiological inhibition of V-ATPase pump activity de-acidifies the vacuole lumen in living yeast cells within minutes. Time-lapse microscopy revealed that de-acidification induces vacuole fusion rather than inhibiting it. Cells expressing mutated V0 subunits that maintain vacuolar acidity were blocked in this fusion. Thus, proton pump activity of the V-ATPase negatively regulates vacuole fusion in vivo. Vacuole fusion in vivo does, however, require physical presence of a fusion-competent V0 sector.
V-ATPase 是一种质子泵，由膜整合的 V0 部分和携带 ATP 酶活性的外围 V1 部分组成。酵母液泡融合的体外研究以及来自蠕虫、苍蝇、斑马鱼和老鼠的证据表明，V0 与 SNARE 机制相互作用以促进膜融合，它促进半融合的诱导，并且这种活性需要 V0 的物理存在而不是其质子泵活性。最近在酵母中的一项体内研究对这些解释提出了挑战，得出的结论是融合仅需要内腔酸化而不是 V0 部分本身。在这里，我们确定了出现这种差异的原因，并对其进行了调和。我们发现，急性药理学或生理学抑制 V-ATPase 泵活性会在数分钟内使活酵母细胞中的液泡内腔去酸化。延时显微镜显示，去酸化会诱导液泡融合，而不是抑制融合。表达维持液泡酸度的突变 V0 亚基的细胞在这种融合中被阻断。因此，V-ATPase 的质子泵活性在体内负调节液泡融合。然而，体内液泡融合确实需要具有融合能力的 V0 部分的物理存在。
