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Title:Phosphatidylinositol 3,5-bisphosphate: A Molecular Switch of Vacuolar Fusion and Fission
Author(s):Guo, Annie; Miner, Gregory; Sullivan, Katie
Contributor(s):Fratti, Rutilio A.
Abstract:Abstract Vacuoles purified from Saccharomyces cerevisiae are a well-studied model for membrane fusion and fission as the machinery is highly conserved throughout eukaryotes. Vacuole membranes undergo cycles of fusion and fission which have distinct mechanisms but are in part controlled by overlapping regulators. Both processes are dependent on proteins, ion concentrations, and lipid composition, highlighting the complex regulation of vacuole homeostasis. Previous studies have shown the lipid PI(3,5)P2 (phosphatidylinositol 3,5-bisphosphate) is a crucial activator of vacuolar fission. PI(3,5)P2 is generated by the PI3P 5-kinase Fab1 . Fab1 is activated in response to osmotic stress leading to a sharp rise in PI(3,5)P2 levels. Increases in PI(3,5)P2 activates the calcium channel Yvc1, causing calcium to efflux from the vacuole. This, along with PI(3,5)P2 activation of Vph1 (a subunit of a vacuolar V-ATPase) results in fragmentation of the vacuole. Here we show that PI(3,5)P2 is a novel inhibitor of vacuolar fusion. Additionally we found PI(3,5)P2 does not prevent fusion by inhibiting priming and trans-SNARE pairing (the early steps of fusion). In order to look at the later steps of fusion, we conducted lipid mixing experiments to measure it’s effects on hemi-fusion, the precursor step to vacuolar fusion. Our results show that PI(3,5)P2 inhibits hemi-fusion, but the exact mechanism is still unclear. We also hypothesized that PI(3,5)P2 acts to inhibit fusion through either the Yvc1 calcium efflux or the Vph1 V-ATPase pathway. Vph1 interactions promote either vacuolar fusion or fission depending on binding partners. We predicted PI(3,5)P2 to disrupt Vph1 fusion complexes while promoting fission complexes. But, experiments using TAP-tagged Vph1 were inconclusive. Also, experiments with Yvc1 knockout yeast retained sensitivity to PI(3,5)P2 - interestingly, the calcium influx pump Pmc1 was found to be enhanced by PI(3,5)P2, which may explain decreased calcium influx.
Issue Date:2018-04
Genre:Conference Poster
Rights Information:Copyright 2018 Annie Guo
Copyright 2018 Gregory Miner
Copyright 2018 Katie Sullivan
Date Available in IDEALS:2018-05-23

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