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Title: Phosphatidylinositol 3, 5‐bisphosphate regulates Ca 2+ transport during yeast vacuolar fusion through the Ca 2+ ATPase Pmc1

The transport of Ca2+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 Ca2+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)P2pools. Ca2+is also released during vacuole fusion upontrans‐SNARE complex assembly, however, its role remains unclear. The effect of PI(3,5)P2on Ca2+flux during fusion was independent of Yvc1. Here, we show that while low levels of PI(3,5)P2were required for Ca2+uptake into the vacuole, increased concentrations abolished Ca2+efflux. This was as shown by the addition of exogenous dioctanoyl PI(3,5)P2or increased endogenous production of by the hyperactivefab1T2250Amutant. In contrast, the lack of PI(3,5)P2on vacuoles from the kinase deadfab1EEEmutant showed delayed and decreased Ca2+uptake. The effects of PI(3,5)P2were linked to the Ca2+pump Pmc1, as its deletion rendered vacuoles resistant to the effects of excess PI(3,5)P2. Experiments with Verapamil inhibited Ca2+uptake when added at the start of the assay, while adding it after Ca2+had been taken up resulted in the rapid expulsion of Ca2+. Vacuoles lacking both Pmc1 and the H+/Ca2+exchanger Vcx1 lacked the ability to take up Ca2+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)P2can modulate which path predominates.

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p. 503-517
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National Science Foundation
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