Vps41 phosphorylation and the Rab Ypt7 control the targeting of the HOPS complex to endosome-vacuole fusion sites.
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.
Cabrera
Margarita
M
Biochemistry Section, Department of Biology, University of Osnabrück, 49076 Osnabrück, Germany.
Ostrowicz
Clemens W
CW
Mari
Muriel
M
LaGrassa
Tracy J
TJ
Reggiori
Fulvio
F
Ungermann
Christian
C
D033963:Adaptor Protein Complex 3 / Q000378:metabolism; D000595:Amino Acid Sequence; D047389:Casein Kinase I / Q000378:metabolism; D021961:Cell Membrane Structures / Q000378:metabolism / Q000648:ultrastructure; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D020690:GTPase-Activating Proteins / Q000378:metabolism; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism* / Q000648:ultrastructure; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
D033963:Adaptor Protein Complex 3; D020690:GTPase-Activating Proteins; D046912:Multiprotein Complexes; D050505:Mutant Proteins; D029701:Saccharomyces cerevisiae Proteins; C083878:VPS41 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D047389:Casein Kinase I; C479667:YCK3 protein, S cerevisiae; C079005:YPT7 protein, S cerevisiae; D020691:rab GTP-Binding Proteins