title: The Mon1-Ccz1 GEF activates the Rab7 GTPase Ypt7 via a longin-fold-Rab interface and association with PI3P-positive membranes.
abstract: To function in fusion and signaling, Rab GTPases need to be converted into their active GTP form. We previously identified the conserved Mon1-Ccz1 complex as the guanine nucleotide exchange factor (GEF) of the yeast Rab7 GTPase Ypt7. To address the possible GEF mechanism, we generated a homology model of the predicted longin domains of Mon1 and Ccz1 using the Rab-binding surface of the TRAPP complex as a template. On the basis of this, we identified mutations in both yeast Mon1 and Ccz1 that block Ypt7 activation, without affecting heterodimer formation and intracellular localization of Mon1 and Ccz1 at endosomes. Strikingly, the activity of the isolated Mon1-Ccz1 complex for Ypt7 is highly stimulated on membranes, and is promoted by the same anionic phospholipids such as phosphatidylinositol-3-phosphate (PI3P), which also support membrane association of the GEF complex. Our data imply that the GEF activity of the Mon1-Ccz1 complex towards Rab7/Ypt7 requires the interface formed by their longin domains and profits strongly from its association with the organelle surface.
authors.affiliation: University of Osnabrück, Department of Biology/Chemistry, Biochemistry section, Barbarastrasse 13, 49076 Osnabrück, Germany.
authors.forename: Margarita
authors.initials: M
authors.lastname: Cabrera
authors.forename: Mirjana
authors.initials: M
authors.lastname: Nordmann
authors.forename: Angela
authors.initials: A
authors.lastname: Perz
authors.forename: David
authors.initials: D
authors.lastname: Schmedt
authors.forename: Andreas
authors.initials: A
authors.lastname: Gerondopoulos
authors.forename: Francis
authors.initials: F
authors.lastname: Barr
authors.forename: Jacob
authors.initials: J
authors.lastname: Piehler
authors.forename: Siegfried
authors.initials: S
authors.lastname: Engelbrecht-Vandré
authors.forename: Christian
authors.initials: C
authors.lastname: Ungermann
mesh_terms: D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; 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 / Q000737:chemistry / Q000378:metabolism*
chemical_list: C441960:Ccz1 protein, S cerevisiae; D020662:Guanine Nucleotide Exchange Factors; C470635:Mon1 protein, S cerevisiae; D018129:Phosphatidylinositol Phosphates; D010718:Phosphatidylserines; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; C055525:phosphatidylinositol 3-phosphate; C079005:YPT7 protein, S cerevisiae; D020691:rab GTP-Binding Proteins
keywords: Endosome; Guanine nucleotide exchange factor; Membrane fusion; Mon1–Ccz1; Rab GTPase