The Rab to Rab switch on endosomes

The Rab5 to Rab7 switch on endosomes co-occurs with the release of the Rab5 GEF Rabex5 from endosomes, which prevents further Rab5 activation [5]. In addition, the Rab5 GAP is recruited to inactivate remaining Rab5 [67]. Recent data suggest that another GAP, TBC1D5, which binds the retromer complex, also controls Rab7 activity 68, 69, 70. This would couple the inactivation of Rab7 closely to recycling events at the level of late endosomes, orchestrated by the retromer complex. In yeast, the transition from Vps21 to Ypt7 requires the endosomal BLOC-1 complex, which directly binds the GAP of Vps21, called Msb3 71, 72, 73. Msb3 also acts on the exocytic Sec4 Rab [74]. Yeast BLOC-1 is homologous to two metazoan complexes, the BLOC-1 complex involved in biogenesis of lysosome-related organelles 75, 76, 77 and the lysosomal BORC complex with a role in lysosomal positioning [78]. It will be important to unravel the exact molecular interplay to clarify which functions of BLOC-1 and BORC are conserved across species.
Membrane Tethering at Early and Late Endosomes Rabs are considered as the main organizers of vesicle tethering, but the fusion of vesicles with organelles depends on tethering complexes and small transmembrane proteins called SNAREs. SNAREs are sufficient to mediate fusion of liposomes, since they catalyze the mixing of the two membrane bilayers. They are required for almost all fusion events occurring within the endomembrane system of eukaryotic alizarin mg 79, 80. Even though Rabs are required in vivo, their function can be bypassed by SNARE overproduction, as shown with purified yeast vacuoles in vitro[81]. However, reconstituted assays with physiological SNARE concentrations 82, 83, and the essential role of Rab5 in mouse development [45] demonstrate that Rab-GTPases determine where fusion occurs. In fact, SNARE-dependent fusion under physiological conditions requires tethering factors, which depend on membrane-localized Rabs 82, 84, 85. Consequently, Rab GEFs and GAPs have a central regulator function as fusion-promoting and -limiting factors [19]. It was recently observed that the Rab7 GEF Mon1-Ccz1 is necessary and sufficient to recruit Ypt7 from the GDI complex to membranes, and, thus, to control the SNARE- and HOPS-tethering complex (discussed below)-dependent fusion of proteoliposomes [86]. This shows that fusion can be controlled by the GEF as the most upstream factor in a fusion cascade (Figure 2C).
Rabs and Tethers Orchestrate SNAREs Efficient membrane fusion depends on tight coupling between Rab-GTPases, tethers, SM proteins, and SNAREs. Rabs, in concert with tethering factors, appear to mark the site of fusion 8, 10. The Rab-recruited tethers then interact with the central fusion machinery, the SNARE proteins. SNAREs are type II membrane proteins (mostly) with a conserved SNARE domain proximal to the transmembrane segment. They have been classified as Q- and R-SNAREs according to a central hydrophilic residue of the otherwise hydrophobic coiled-coil interface [114]. Individual SNAREs can zipper into tightly folded four-helical bundles in a folding reaction that starts along the SNARE domain at the distal N-terminal domain and proceeds to the membrane proximal C-terminal domain. This folding reaction brings apposing membranes into close proximity. In vivo, this SNARE assembly is chaperoned by SM proteins, which directly bind to the SNARE domains of the involved proteins 80, 101. SM proteins either associate with tethers or are, as already described for HOPS and CORVET, an integral part of the tethering complex. Regulated exocytosis also includes Ca2+-sensing proteins, such as synaptotagmins, thus coupling Ca2+ release with rapid fusion [85]. Once the bilayers have mixed, the AAA-ATPase N-ethylmaleimide-sensitive factor (NSF) and its cofactor α-SNAP bind SNAREs and untangle them for further use [85].
Concluding Remarks and Future Directions