Margarita Cabrera

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.

Phosphorylation of a membrane curvature–sensing motif switches function of the HOPS subunit Vps41 in membrane tethering

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.

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.

Functional Separation of Endosomal Fusion Factors and the Class C Core Vacuole/Endosome Tethering (CORVET) Complex in Endosome Biogenesis

Background: The interdependence of the endosomal fusion factors CORVET and Vac1 has not been addressed. Results: CORVET can be separated from other endocytic fusion factors on the basis of ultrastructure, localization, and trafficking. Conclusion: CORVET acts independently of the Vac1 tether and requires activated Rab5 homologs for localization. Significance: Our data reveal a unique role of CORVET in sorting of biosynthetic cargo to the vacuole. Transport along the endolysosomal system requires multiple fusion events at early and late endosomes. Deletion of several endosomal fusion factors, including the Vac1 tether and the Class C core vacuole/endosome tethering (CORVET) complex-specific subunits Vps3 and Vps8, results in a class D vps phenotype. As these mutants have an apparently similar defect in endosomal transport, we asked whether CORVET and Vac1 could still act in distinct tethering reactions. Our data reveal that CORVET mutants can be rescued by Vac1 overexpression in the endocytic pathway but not in CPY or Cps1 sorting to the vacuole. Moreover, when we compared the ultrastructure, CORVET mutants were most similar to deletions of the Rab Vps21 and its guanine nucleotide exchange factor Vps9 and different from vac1 deletion, indicating separate functions. Likewise, CORVET still localized to endosomes even in the absence of Vac1, whereas Vac1 localization became diffuse in CORVET mutants. Importantly, CORVET localization requires the Rab5 homologs Vps21 and Ypt52, whereas Vac1 localization is strictly Vps21-dependent. In this context, we also uncover that Muk1 can compensate for loss of Vps9 in CORVET localization, indicating that two Rab5 guanine nucleotide exchange factors operate in the endocytic pathway. Overall, our study reveals a unique role of CORVET in the sorting of biosynthetic cargo to the vacuole/lysosome.

Guanine nucleotide exchange factors (GEFs) have a critical but not exclusive role in organelle localization of Rab GTPases

Background: Rab localization has been ascribed to guanine nucleotide exchange factor (GEF) localization. Results: GEF deletions result in mislocalization of Rabs to other membranes, which can be bypassed by a Rab mutant. Conclusion: GEFs are critical for Rab localization, but Rabs also have a GEF-independent ability to localize correctly. Significance: Our data reveal that both GEFs and Rabs contribute to Rab localization in cells. Membrane fusion at eukaryotic organelles is initiated by Rab GTPases and tethering factors. Rabs in their GDP-bound form are kept soluble in the cytoplasm by the GDP dissociation inhibitor (GDI) chaperone. Guanine nucleotide exchange factors (GEFs) are found at organelles and are critical for Rab function. Here, we surveyed the overall role of GEFs in Rab localization. We show that GEFs, but none of the proposed GDI displacement factors, are essential for the correct membrane localization of yeast Rabs. In the absence of the GEF, Rabs lost their primary localization to the target organelle. Several Rabs, such as vacuolar Ypt7, were found at the endoplasmic reticulum and thus were still membrane-bound. Surprisingly, a Ypt7 mutant that undergoes facilitated nucleotide exchange localized to vacuoles independently of its GEF Mon1-Ccz1 and rescued vacuole morphology. In contrast, wild-type Ypt7 required its GEF for localization and to counteract the extraction by GDI. Our data agree with the emerging model that GEFs are critical for Rab localization but raise the possibility that additional factors can contribute to this process.

Purification and In Vitro Analysis of Yeast Vacuoles

The purification of eukaryotic organelles is a prerequisite for the detailed analysis of protein sorting, localization and translocation, membrane fusion and vesicle budding. Yeast vacuoles receive cargo from the exocytic, endocytic, and autophagic pathways and hence represent an excellent model system for the study of organelle biogenesis and protein sorting. Yeast vacuoles undergo fission and fusion in vivo, events that can be monitored in vitro by an assay that employs purified vacuoles from two tester strains. Here, we describe the methodology of yeast vacuole purification, and provide protocols for the detailed analysis of the fusion reaction. We also include methods to analyze protein dynamics on yeast vacuoles and the controls required to ensure their reliability.

The BLOC-1 complex promotes endosomal maturation by recruiting the Rab5 GTPase-activating protein Msb3.

Yeast BLOC-1 acts as both a Rab5–Vps21 effector and an adapter for the Rab-GAP Msb3 to promote endosomal maturation.

The vacuolar V1/V0-ATPase is involved in the release of the HOPS subunit Vps41 from vacuoles, vacuole fragmentation and fusion

At yeast vacuoles, phosphorylation of the HOPS subunit Vps41 depends on the Yck3 kinase. In a screen for mutants that mimic the yck3Δ phenotype, in which Vps41 accumulates in vacuolar dots, we observed that mutants in the V0‐part of the V0/V1‐ATPase, in particular in vma16Δ, also accumulate Vps41. This accumulation is not due to a phosphorylation defect, but to reduced release of Vps41 from vma16Δ vacuoles. One reason could be a connection to vacuole fission, which is blocked in V‐ATPase mutants. Vacuole fusion is not impaired between vacuoles lacking the V0‐subunits Vma16 or Vma6 and wild‐type vacuoles, whereas fusion between mutant vacuoles is reduced. Our data suggest a connection between vacuole biogenesis and membrane fusion.

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

Recruitment and activation of the late endosomal Rab Ypt7 require the GEF Mon1-Ccz1. Association of Mon1 with vacuoles depends on the lipid PI3P, and Mon1 is phosphorylated by the casein kinase Yck3. Phospho-Mon1 is subsequently released from vacuoles as part of a putative recycling mechanism.

Guiding Endosomal Maturation

In the endocytic pathway, early endosomes are converted into late endosomes by exchange of their associated Rab GTPases. In this issue, Poteryaev et al. (2010) identify the SAND-1/Mon1 protein as a switch that shuts off the recruitment of one Rab (Rab5) and facilitates the activation of the next (Rab7).