Background: Pah1p is a phosphatidic acid phosphatase that generates diacylglycerol. Results: Deletion of PAH1 alters the sorting of fusion factors to the vacuole and inhibits fusion. Conclusion: Conversion of phosphatidic acid to diacylglycerol is integral to vacuole homeostasis. Significance: This is the first report that links a Lipin 1 homologue to the recruitment and activation of a Rab GTPase or the sorting of SNARE proteins.
Maturation of organelles in the endolysosomal pathway requires exchange of the early endosomal GTPase Rab5/Vps21 for the late endosomal Rab7/Ypt7. The Rab exchange depends on the guanine nucleotide exchange factor activity of the Mon1-Ccz1 heterodimer for Ypt7. Here we investigate vacuole binding and recycling of Mon1-Ccz1. We find that Mon1-Ccz1 is absent on vacuoles lacking the phosphatidic acid phosphatase Pah1, which also lack Ypt7, the phosphatidylinositol 3-kinase Vps34, and the lipid phosphatidylinositol 3-phosphate (PI3P). Interaction of Mon1-Ccz1 with wild-type vacuoles requires PI3P, as shown in competition experiments. We also find that Mon1 is released from vacuoles during the fusion reaction and its release requires its phosphorylation by the type 1 casein kinase Yck3. In contrast, Mon1 is retained on vacuoles lacking Yck3 or when Mon1 phosphorylation sites are mutated. Phosphorylation and release of Mon1 is restored with addition of recombinant Yck3. Together the results show that Mon1 is recruited to endosomes and vacuoles by PI3P and, likely after activating Ypt7, is phosphorylated and released from vacuoles for recycling.
Yeast vacuole fusion requires the formation of SNARE bundles between membranes. Although the function of vacuolar SNAREs is controlled in part by regulatory lipids, the exact role of the membrane in regulating fusion remains unclear. Because SNAREs are membrane-anchored and transmit the force required for fusion to the bilayer, we hypothesized that the lipid composition and curvature of the membrane aid in controlling fusion. Here, we examined the effect of altering membrane fluidity and curvature on the functionality of fusion-incompetent SNARE mutants that are thought to generate insufficient force to trigger the hemifusion-fusion transition. The hemifusion-fusion transition was inhibited by disrupting the 3Q:1R stoichiometry of SNARE bundles with the mutant SNARE Vam7pQ283R. Similarly, replacing the transmembrane domain of the syntaxin homolog Vam3p with a lipid anchor allowed hemifusion, but not content mixing. Hemifusion-stalled reactions containing either of the SNARE mutants were stimulated to fuse with chlorpromazine, an amphipathic molecule that alters membrane fluidity and curvature. The activity of mutant SNAREs was also rescued by the overexpression of SNAREs, thus multiplying the force transferred to the membrane. Thus, we conclude that either increasing membrane fluidity, or multiplying SNARE-generated energy restored the fusogenicity of mutant SNAREs that are stalled at hemifusion. We also found that regulatory lipids differentially modulated the complex formation of wild-type SNAREs. Together, these data indicate that the physical properties and the lipid composition of the membrane affect the function of SNAREs in promoting the hemifusion-fusion transition.
SummaryHomotypic vacuole fusion requires SNAREs, the Rab Ypt7p, the tethering complex HOPS, regulatory lipids and actin. In Saccharomyces cerevisiae, actin functions at two stages of vacuole fusion. Pre-existing actin filaments are depolymerized to allow docking and assembly of the vertex ring (a microdomain enriched in proteins and lipids that mediate fusion). Actin is then polymerized late in the pathway to aid fusion. Here, we report that the fusion machinery regulates the accumulation of actin at the vertex ring. Using Cy3-labeled yeast actin to track its dynamics, we found that its vertex enrichment was abolished when actin monomers were stabilized by latrunculin-B, independent of the extent of incorporation. By contrast, stabilization of filamentous actin with jasplakinolide markedly augmented actin vertex enrichment. Importantly, agents that inhibit SNAREs, Ypt7p and HOPS inhibited the vertex enrichment of actin, demonstrating that the cytoskeleton and the fusion machinery are interdependently regulated. Actin mobilization was also inhibited by ligating ergosterol and PtdIns(3)P, whereas the ligation or modification of PtdIns(4,5)P 2 augmented the vertex enrichment of actin. The proteins and lipids that regulated actin mobilization to the vertex did not affect the total incorporation of Cy3-actin, indicating that actin mobilization and polymerization activities can be dissociated during membrane fusion.
Background: Ycf1p is a ABCC transporter that is localized to the vacuole and that was initially characterized as a cadmium transporter. Results: Deletion of YCF1 inhibits vacuole fusion in part by excluding the soluble SNARE Vam7p.
Conclusion:The vacuole fusion machinery requires Ycf1p function for efficient fusion. Significance: This is the first report that an ABCC protein affects fusion through the recruitment of a SNARE.
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