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)P₂ pools. Ca²⁺ is also released during vacuole fusion upon trans-SNARE complex assembly, however, its role remains unclear. The effect of PI(3,5)P₂ on Ca²⁺ flux during fusion was independent of Yvc1. Here, we show that while low levels of PI(3,5)P₂ were 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)P₂ or increased endogenous production of by the hyperactive fab1^T2250A mutant. In contrast, the lack of PI(3,5)P₂ on vacuoles from the kinase dead fab1^EEE mutant showed delayed and decreased Ca²⁺ uptake. The effects of PI(3,5)P₂ were 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)P₂ can modulate which path predominates.

• PMID: 32388897
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Journal Article | Research Support, N.I.H., Extramural | Research Support, U.S. Gov't, Non-P.H.S.

Authors

Miner GE, Sullivan KD, Zhang C, Rivera-Kohr D, Guo A, Hurst LR, Ellis EC, Starr ML, Jones BC, Fratti RA

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