Phosphatidylinositol 3,5-Bisphosphate regulates Ca 2+ Transport During Yeast Vacuolar Fusion through the Ca 2+ ATPase Pmc1.

The transport of Ca(2+)across membranes precedes the fusion and fission of various lipid bilayers. Yeast vacuoles under hyperosmotic stress become fragmented through fission events that requires the release of Ca(2+)stores through the TRP channel Yvc1. This requires the phosphorylation of phosphatidylinositol-3-phosphate (PI3P) by the PI3P-5-kinase Fab1 to produce transient PI(3,5)P(2)pools. Ca(2+)is also released during vacuole fusion upontrans-SNARE complex assembly, however, its role remains unclear. The effect of PI(3,5)P(2)on Ca(2+)flux during fusion was independent of Yvc1. Here, we show that while low levels of PI(3,5)P(2)were required for Ca(2+)uptake into the vacuole, increased concentrations abolished Ca(2+)efflux. This was as shown by the addition of exogenous dioctanoyl PI(3,5)P(2)or increased endogenous production of by the hyperactivefab1(T2250A)mutant. In contrast, the lack of PI(3,5)P(2)on vacuoles from the kinase deadfab1(EEE)mutant showed delayed and decreased Ca(2+)uptake. The effects of PI(3,5)P(2)were linked to the Ca(2+)pump Pmc1, as its deletion rendered vacuoles resistant to the effects of excess PI(3,5)P-2. Experiments with Verapamil inhibited Ca(2+)uptake when added at the start of the assay, while adding it after Ca(2+)had been taken up resulted in the rapid expulsion of Ca2+. Vacuoles lacking both Pmc1 and the H+/Ca(2+)exchanger Vcx1 lacked the ability to take up Ca(2+)and instead expelled it upon the addition of ATP. Together these data suggest that a balance of efflux and uptake compete during the fusion pathway and that the levels of PI(3,5)P(2)can modulate which path predominates.