Docked vacuoles are believed to undergo rapid lipid mixing during hemifusion and then a slow, rate-limiting completion of fusion and mixing of lumenal contents. Previous genomic analysis has suggested that Bem1p, a scaffold protein critical for cell polarity, may support vacuole fusion. We now report that bem1Delta strains have fragmented vacuoles (vps class B and C). During in vitro fusion reactions, vacuoles from bem1Delta strains showed a strong reduction in the rate of lipid mixing when compared with vacuoles from the BEM1 parent. The reduction in the overall rate of fusion with bem1Delta vacuoles was modest, consistent with lipid mixing as a non-rate-limiting step in the pathway. Although the fusion of either BEM1 (wild-type) or bem1Delta vacuoles is stimulated by recombinant Bem1p, the lipid mixing of docked bem1Delta vacuoles is highly dependent on rBem1p under certain reaction conditions. Bem1p-stimulated lipid mixing is blocked by well characterized fusion inhibitors including lipid ligands and antibodies to Ypt7p, Vps33p, and Vam3p. Although full-length Bem1p is required for maximal stimulation, a truncation mutant comprising the SH3 domains and the Phox homology (PX) domain retains modest stimulatory activity. In contrast to an earlier report (Han, B. K., Bogomolnaya, L. M., Totten, J. M., Blank, H. M., Dangott, L. J., and Polymenis, M. (2005) Genes Dev. 19, 2606-2618), we did not find phosphorylation of Bem1p at Ser-72 to be required for Bem1p-stimulated fusion. Taken together, Bem1p is a positive regulator of lipid mixing during vacuole hemifusion and fusion.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|