title: Dynamic association of the PI3P-interacting Mon1-Ccz1 GEF with vacuoles is controlled through its phosphorylation by the type 1 casein kinase Yck3.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
authors.forename: Gus
authors.initials: G
authors.lastname: Lawrence
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Brown
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
authors.forename: Blake A
authors.initials: BA
authors.lastname: Flood
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
authors.forename: Surya
authors.initials: S
authors.lastname: Karunakaran
authors.affiliation: Biochemistry Section, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany.
authors.forename: Margarita
authors.initials: M
authors.lastname: Cabrera
authors.affiliation: Biochemistry Section, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany.
authors.forename: Mirjana
authors.initials: M
authors.lastname: Nordmann
authors.affiliation: Biochemistry Section, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Ungermann
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801 rfratti@illinois.edu.
authors.forename: Rutilio A
authors.initials: RA
authors.lastname: Fratti
mesh_terms: D047389:Casein Kinase I / Q000235:genetics / Q000378:metabolism*; D058546:Class III Phosphatidylinositol 3-Kinases / Q000235:genetics; D011992:Endosomes / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D010711:Phosphatidate Phosphatase / Q000235:genetics; D018129:Phosphatidylinositol Phosphates / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins
chemical_list: C441960:Ccz1 protein, S cerevisiae; D020662:Guanine Nucleotide Exchange Factors; C470635:Mon1 protein, S cerevisiae; D018129:Phosphatidylinositol Phosphates; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; C055525:phosphatidylinositol 3-phosphate; D000090802:rab7 GTP-Binding Proteins; D058546:Class III Phosphatidylinositol 3-Kinases; D047389:Casein Kinase I; C479667:YCK3 protein, S cerevisiae; C079831:PAH1 protein, S cerevisiae; D010711:Phosphatidate Phosphatase; C086866:VPS21 protein, S cerevisiae; C079005:YPT7 protein, S cerevisiae; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins