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.

Emerging evidence suggests that contact sites between different organelles form central hubs in the coordination of cellular physiology. Although recent work has emphasized the crucial role of the endoplasmic reticulum in interorganellar crosstalk, the cooperative behavior of other organelles is largely unexplored. Here, we identify a contact site named vCLAMP (vacuole and mitochondria patch) that integrates mitochondria with the lysosome-like vacuole and thus the endocytic pathway. vCLAMPs depend on the vacuolar HOPS tethering complex subunit Vps39/Vam6 and the Rab GTPase Ypt7, which also participate in membrane fusion at the vacuole. Intriguingly, vCLAMPs are located proximal to the ER-mitochondria encounter structure (ERMES) complexes, and an increase in vCLAMPs can rescue the growth defect of ERMES mutants. Importantly, the persistence of vCLAMPs is regulated by phosphorylation of Vps39 and is strongly reduced during respiratory growth. The identification of this organelle contact site reveals a physical and metabolic interconnection between the endocytic pathway and mitochondria.

An AP-3–binding site required for vesicle–vacuole fusion is masked when Vps41 is associated with highly curved membranes, such as endosomes, but is exposed at membranes with lower curvature, such as vacuoles, because of phosphorylation of the membrane-binding motif.

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.

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 Rabbinding 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.