1. Vps41 Phosphorylation and the Rab Ypt7 Control the Targeting of the HOPS Complex to Endosome–Vacuole Fusion Sites
- Author
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Clemens W. Ostrowicz, Tracy J. LaGrassa, Muriel Mari, Christian Ungermann, Margarita Cabrera, and Fulvio Reggiori
- Subjects
Saccharomyces cerevisiae Proteins ,Mutation/genetics ,Endosome ,Adaptor Protein Complex 3 ,Saccharomyces cerevisiae/cytology ,Molecular Sequence Data ,Vesicular Transport Proteins ,GTPase-Activating Proteins/metabolism ,Vacuole fusion ,Vacuole ,Endosomes ,Saccharomyces cerevisiae ,Biology ,Cell Membrane Structures ,Membrane Fusion ,Mutant Proteins/metabolism ,Vesicular Transport Proteins/chemistry ,Multiprotein Complexes/metabolism ,rab GTP-Binding Proteins/metabolism ,Amino Acid Sequence ,Phosphorylation ,Molecular Biology ,Vacuolar HOPS complex ,Casein Kinase I ,Vacuoles/metabolism ,GTPase-Activating Proteins ,Adaptor Protein Complex 3/metabolism ,Lipid bilayer fusion ,Endosomes/metabolism ,Cell Biology ,Articles ,Cell biology ,Protein Transport ,Cell Membrane Structures/metabolism ,rab GTP-Binding Proteins ,Multiprotein Complexes ,Saccharomyces cerevisiae Proteins/chemistry ,Mutation ,Vacuoles ,Mutant Proteins ,Casein Kinase I/metabolism ,Rab ,Casein kinase 1 - Abstract
Membrane fusion depends on multisubunit tethering factors such as the vacuolar HOPS complex. We previously showed that the vacuolar casein kinase Yck3 regulates vacuole biogenesis via phosphorylation of the HOPS subunit Vps41. Here, we link the identified Vps41 phosphorylation site to HOPS function at the endosome–vacuole fusion site. The nonphosphorylated Vps41 mutant (Vps41 S-A) accumulates together with other HOPS subunits on punctate structures proximal to the vacuole that expand in a class E mutant background and that correspond to in vivo fusion sites. Ultrastructural analysis of this mutant confirmed the presence of tubular endosomal structures close to the vacuole. In contrast, Vps41 with a phosphomimetic mutation (Vps41 S-D) is mislocalized and leads to multilobed vacuoles, indicative of a fusion defect. These two phenotypes can be rescued by overproduction of the vacuolar Rab Ypt7, revealing that both Ypt7 and Yck3-mediated phosphorylation modulate the Vps41 localization to the endosome–vacuole junction. Our data suggest that Vps41 phosphorylation fine-tunes the organization of vacuole fusion sites and provide evidence for a fusion “hot spot” on the vacuole limiting membrane.
- Published
- 2009
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