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Yvc1 acts as a Novel Suppressor of Vacuolar Membrane Fusion

Yeast vacuoles maintain homeostasis in part by balancing membrane fission and fusion; however the regulation of these events is not fully understood. Phosphoinositides (PIs), and the proteins they interact with, are crucial regulators of both fusion and fission. Our work has shown phosphoinositides are essential for membrane fusion, specifically phosphoinositide 3‐phosphate (PI3P), PI4P, and PI(4,5)P2. Comparatively the phosphoinositide PI(3,5)P2 is relatively scarce and uncharacterized in membrane fusion, however others have demonstrated its requirement in osmotic shock induced membrane fission. Creation of PI(3,5)P2 during osmotic shock is coupled with a release of Ca2+ from the vacuole into the cytosol. This Ca2+ release is thought to be caused by a direct interaction of PI(3,5)P2 with the yeast TRP Ca2+ channel (Yvc1). Since Ca2+ release also plays a role in membrane fusion, we sought to determine whether Yvc1 or its activation by PI(3,5)P2 is involved in membrane fusion as well as fission. Utilizing isolated yeast vacuoles we found that the absence of Yvc1 caused a significant increase in membrane fusion. Our data suggest that the augmented fusion was due to elevated levels of PI3P on the vacuole leading to subsequent increased recruitment of fusion machinery. Specifically, we saw enrichment of the soluble SNARE Vam7, subunits of the HOPS tethering complex, as well as the Rab GTPase Ypt7 and its heterodimeric guanine exchange factor Mon1‐Ccz1. These proteins act as components of the tethering machinery and we found that their enrichment in yvc1D vacuoles led to enhanced trans‐SNARE pairing. The increase in SNARE complexes was not due to enhanced rates of priming, suggesting that the change was due to increased vacuole tethering linked to the increase in Mon1‐Ccz1, Ypt7 and HOPS. Taken together our data suggests Yvc1 acts as a novel suppressor of vacuole membrane fusion through its effects on phosphoinositide levels and recruitment of tethering factors.