Phosphoinositides (PIs) regulate a myriad of cellular functions including membrane fusion, as exemplified by the yeast vacuole, which uses various PIs at different stages of fusion. In light of this, the effect of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) on vacuole fusion remains unknown. PI(3,5)P2 is made by the PI3P 5-kinase Fab1 and has been characterized as a regulator of vacuole fission during hyperosmotic shock, where it interacts with the TRP Ca2+ channel Yvc1. Here we demonstrate that exogenously added dioctanoyl (C8) PI(3,5)P2 abolishes homotypic vacuole fusion. This effect was not linked to Yvc1, as fusion was equally affected using yvc1Δ vacuoles. Thus, the effects of C8-PI(3,5)P2 on fusion and fission operate through distinct mechanisms. Further testing showed that C8-PI(3,5)P2 inhibited vacuole fusion after trans-SNARE pairing. Although SNARE complex formation was unaffected, we found that C8-PI(3,5)P2 blocked outer leaflet lipid mixing. Overproduction of endogenous PI(3,5)P2 by the fab1T2250A hyperactive kinase mutant also inhibited the lipid mixing stage, bolstering the model in which PI(3,5)P2 inhibits fusion when present at elevated levels. Taken together, this work identifies a novel function for PI(3,5)P2 as a regulator of vacuolar fusion. Moreover, it suggests that this lipid acts as a molecular switch between fission and fusion.

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 fab1T2250A mutant. In contrast, the lack of PI(3,5)P2 on vacuoles from the kinase dead fab1EEE 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.

The accumulation of copper in organisms can lead to altered functions of various pathways and become cytotoxic through the generation of reactive oxygen species. In yeast, cytotoxic metals such as Hg+, Cd2+ and Cu2+ are transported into the lumen of the vacuole through various pumps. Copper ions are initially transported into the cell by the copper transporter Ctr1 at the plasma membrane and sequestered by chaperones and other factors to prevent cellular damage by free cations. Excess copper ions can subsequently be transported into the vacuole lumen by an unknown mechanism. Transport across membranes requires the reduction of Cu2+ to Cu+. Labile copper ions can interact with membranes to alter fluidity, lateral phase separation and fusion. Here we found that CuCl2 potently inhibited vacuole fusion by blocking SNARE pairing. This was accompanied by the inhibition of V‐ATPase H+ pumping. Deletion of the vacuolar reductase Fre6 had no effect on the inhibition of fusion by copper. This suggests that Cu2+ is responsible for the inhibition of vacuole fusion and V‐ATPase function. This notion is supported by the differential effects of chelators. The Cu2+‐specific chelator triethylenetetramine rescued fusion, whereas the Cu+‐specific chelator bathocuproine disulfonate had no effect on the inhibited fusion.

Running title: PI(3,5)P 2 and Vacuolar Ca 2+ Flux Abbreviations C8, dioctanoyl; PA, phosphatidic acid; PI3P, phosphatidylinositol 3-phosphate; PI(3,5)P 2 , phosphatidylinositol 3,5-bisphospahte; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor. Summary:During osmotic stress PI(3,5)P 2 triggers Ca 2+ release from vacuoles. Here we show PI(3,5)P 2 stimulates Ca 2+ uptake by vacuoles during fusion, illustrating that it has a dual role in Ca 2+ transport.G.E. Miner,5)P 2 and Vacuolar Ca 2+ Transport 2 ABSTRACT The transport of Ca 2+ across membranes precedes the fusion and fission of various lipid bilayers. Yeast vacuoles during hyperosmotic shock become fragmented through fission events that require Ca 2+ efflux of their luminal stores through the TRP channel Yvc1. This requires the production of the lipid PI(3,5)P 2 by Fab1. Ca 2+ is also released during vacuole fusion upon trans-SNARE complex assembly, however, the role of PI(3,5)P 2 remains unclear. Here we demonstrate that elevated PI(3,5)P 2 levels abolish Ca 2+ efflux during fusion, indicating that PI(3,5)P 2 has opposing effects on Ca 2+ transport in fission versus fusion. Notably, Ca 2+ efflux was enhanced when PI(3,5)P 2 levels were reduced. Importantly, the effect of PI(3,5)P 2 on Ca 2+ flux was independent of Yvc1. Rather, the effect was dependent on the Ca 2+ pump Pmc1. Vacuoles lacking Pmc1 were resistant to the effects of PI(3,5)P 2 , while those lacking Yvc1 remained sensitive. Furthermore altering PI(3,5)P 2 levels affects the interactions of Pmc1 with the V o component Vph1 and the R-SNARE Nyv1. We now propose a model in which elevated PI(3,5)P 2 activates continued Pmc1 function to prevent the accumulation of released extraluminal Ca 2+ .

Using confocal Raman microspectroscopy, we derive parameters for bilayer water transport across an isolated nanoliter aqueous droplet pair. For a bilayer formed with two osmotically imbalanced and adherent nanoliter aqueous droplets in a surrounding oil solvent, a droplet interface bilayer (DIB), the water permeability coefficient across the lipid bilayer was determined from monitoring the Raman scattering from the C[triple bond, length as m-dash]N stretching mode of KFe(CN) as a measure of water uptake into the swelling droplet of a DIB pair. We also derive passive diffusional permeability coefficient for DO transport across a droplet bilayer using O-D Raman signal. This method provides a significant methodological advance in determining water permeability coefficients in a convenient and reliable way.