The Mon1-Ccz1 complex is the GEF of the late endosomal Rab7 homolog Ypt7.

Rab GTPases coordinate membrane fusion reactions [1]. Rab-GDP requires a guanine nucleotide exchange factor (GEF) for its conversion to the active GTP form. It then binds to effectors such as multimeric tethering complexes and supports fusion [2]. GTPase-activating proteins (GAPs) promote GTP hydrolysis to inactivate the Rab. GEFs are thus critical activators of fusion reactions [3, 4]. The Rab GEF family is diverse, ranging from multimeric complexes [5] to monomeric GEFs [6-9]. At the late endosome, Rab7 activation is critical for endosomal maturation. The yeast Rab7 homolog Ypt7 binds to the homotypic fusion and protein sorting (HOPS) complex [10, 11]. Its subunit Vps39/Vam6 has been proposed as a GEF for Ypt7 [12] and the Rag GTPase Gtr1 [13], but other genetic evidence has implicated the endosomal protein Ccz1 as a GEF for Ypt7 [14]. Ccz1 and its binding partner Mon1 have been linked to endosomal transport and maturation [15-20]. We now provide evidence that the dimeric Mon1-Ccz1 complex is the Rab7/Ypt7 GEF. The Mon1-Ccz1 complex, but neither protein alone, counteracts GAP function in vivo, rescues in vitro fusion of vacuoles carrying Ypt7-GDP, and promotes nucleotide exchange on Ypt7 independently of Vps39/HOPS. Our data indicate that the Mon1-Ccz1 complex triggers endosomal maturation by activating Ypt7 on late endosomes.

Nordmann M ，Cabrera M ，Perz A ，Bröcker C ，Ostrowicz C ，Engelbrecht-Vandré S ，Ungermann C ... -  《-》

The Mon1-Ccz1 GEF activates the Rab7 GTPase Ypt7 via a longin-fold-Rab interface and association with PI3P-positive membranes.

Cabrera M ，Nordmann M ，Perz A ，Schmedt D ，Gerondopoulos A ，Barr F ，Piehler J ，Engelbrecht-Vandré S ，Ungermann C ... -  《-》

Identification of a Rab GTPase-activating protein cascade that controls recycling of the Rab5 GTPase Vps21 from the vacuole.

Transport within the endocytic pathway depends on a consecutive function of the endosomal Rab5 and the late endosomal/lysosomal Rab7 GTPases to promote membrane recycling and fusion in the context of endosomal maturation. We previously identified the hexameric BLOC-1 complex as an effector of the yeast Rab5 Vps21, which also recruits the GTPase-activating protein (GAP) Msb3. This raises the question of when Vps21 is inactivated on endosomes. We provide evidence for a Rab cascade in which activation of the Rab7 homologue Ypt7 triggers inactivation of Vps21. We find that the guanine nucleotide exchange factor (GEF) of Ypt7 (the Mon1-Ccz1 complex) and BLOC-1 both localize to the same endosomes. Overexpression of Mon1-Ccz1, which generates additional Ypt7-GTP, or overexpression of activated Ypt7 promotes relocalization of Vps21 from endosomes to the endoplasmic reticulum (ER), which is indicative of Vps21 inactivation. This ER relocalization is prevented by loss of either BLOC-1 or Msb3, but it also occurs in mutants lacking endosome-vacuole fusion machinery such as the HOPS tethering complex, an effector of Ypt7. Importantly, BLOC-1 interacts with the HOPS on vacuoles, suggesting a direct Ypt7-dependent cross-talk. These data indicate that efficient Vps21 recycling requires both Ypt7 and endosome-vacuole fusion, thus suggesting extended control of a GAP cascade beyond Rab interactions.

Rana M ，Lachmann J ，Ungermann C 《-》

Dynamic association of the PI3P-interacting Mon1-Ccz1 GEF with vacuoles is controlled through its phosphorylation by the type 1 casein kinase Yck3.

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.

Lawrence G ，Brown CC ，Flood BA ，Karunakaran S ，Cabrera M ，Nordmann M ，Ungermann C ，Fratti RA ... -  《-》

A guanine nucleotide exchange factor (GEF) limits Rab GTPase-driven membrane fusion.

The identity of organelles in the endomembrane system of any eukaryotic cell critically depends on the correctly localized Rab GTPase, which binds effectors and thus promotes membrane remodeling or fusion. However, it is still unresolved which factors are required and therefore define the localization of the correct fusion machinery. Using SNARE-decorated proteoliposomes that cannot fuse on their own, we now demonstrate that full fusion activity can be achieved by just four soluble factors: a soluble SNARE (Vam7), a guanine nucleotide exchange factor (GEF, Mon1-Ccz1), a Rab-GDP dissociation inhibitor (GDI) complex (prenylated Ypt7-GDI), and a Rab effector complex (HOPS). Our findings reveal that the GEF Mon1-Ccz1 is necessary and sufficient for stabilizing prenylated Ypt7 on membranes. HOPS binding to Ypt7-GTP then drives SNARE-mediated fusion, which is fully GTP-dependent. We conclude that an entire fusion cascade can be controlled by a GEF.

Langemeyer L ，Perz A ，Kümmel D ，Ungermann C ... -  《-》