title: The Msb3/Gyp3 GAP controls the activity of the Rab GTPases Vps21 and Ypt7 at endosomes and vacuoles.
abstract: Fusion of organelles in the endomembrane system depends on Rab GTPases that interact with tethering factors before lipid bilayer mixing. In yeast, the Rab5 GTPase Vps21 controls fusion and membrane dynamics between early and late endosomes. Here we identify Msb3/Gyp3 as a specific Vps21 GTPase-activating protein (GAP). Loss of Msb3 results in an accumulation of Vps21 and one of its effectors Vps8, a subunit of the CORVET complex, at the vacuole membrane in vivo. In agreement, Msb3 forms a specific transition complex with Vps21, has the highest activity of all recombinant GAPs for Vps21 in vitro, and is found at vacuoles despite its predominant localization to bud tips and bud necks at the plasma membrane. Surprisingly, Msb3 also inhibits vacuole fusion, which can be rescued by the Ypt7 GDP-GTP exchange factor (GEF), the Mon1-Ccz1 complex. Consistently, msb3 vacuoles fuse more efficiently than wild-type vacuoles in vitro, suggesting that GAP can also act on Ypt7. Our data indicate that GAPs such as Msb3 can act on multiple substrates in vivo at both ends of a trafficking pathway. This ensures specificity of the subsequent GEF-mediated activation of the Rab that initiates the next transport event.
authors.affiliation: Biochemistry Section, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Lachmann
authors.forename: Francis A
authors.initials: FA
authors.lastname: Barr
authors.forename: Christian
authors.initials: C
authors.lastname: Ungermann
mesh_terms: D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004705:Endocytosis; D011992:Endosomes / Q000201:enzymology* / Q000378:metabolism; D004789:Enzyme Activation; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D025941:Protein Interaction Mapping; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology* / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D020690:GTPase-Activating Proteins; C540987:MSB3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C103153:VPS8 protein, S cerevisiae; D033921:Vesicular Transport Proteins; C086866:VPS21 protein, S cerevisiae; C079005:YPT7 protein, S cerevisiae; D020691:rab GTP-Binding Proteins