br Membrane Fusion Requires Rab GTPases

Membrane Fusion Requires Rab-GTPases, Tethers, and SNAREs
Regulation and Function of Rab5 Following endocytosis in metazoan cells, the Rab5-specific GEF Rabex5 binds to ubiquitinated cargo proteins, such as cell surface receptors or metabolite transporters, and subsequently activates Rab5 on endocytic vesicles 27, 28 (Figure 2B). Likewise, the homologous yeast Vps9 interacts with ubiquitin on endosomes 29, 30, 31, 32. In mammalian cells, Rabex5 forms a complex with a Rab5 effector, Rabaptin 5 [33]. This suggests that Rab5-GTP on membranes concentrates the Rab5-GEF close by, thus triggering the formation of a Rab5 domain on endosomes. Recent analyses of Rabaptin 5 suggest that this model needs refinement because Rabaptin 5 is also a Rab4 effector [34]. Rab4, a member of the Rab11-family, is involved in endocytic recycling pathways 11, 35. Recruitment of the Rabaptin5-Rabex5 complex by Rab4 could thereby couple the Rab5 activation loop closely to recycling pathways between early endosomes and the plasma membrane. Metazoan Rab5-GTP recruits a number of effectors on the endosomal membrane (Figure 1). One prominent member is early endosome antigen 1 (EEA1), a tethering protein required for fusion of endocytic vesicles with early endosomes [36]. Another Rab5 effector, the CORVET complex, is involved in tethering and fusion of a subset of endosomes in metazoan ARCA Cy5 EGFP mg that are positive for the APPL1 transcription factor 37, 38. CORVET was initially identified in yeast, where it requires the Rab5 homolog Vps21 for localization and function 39, 40, 41. Rab5 also recruits and activates the PI-3-kinase Vps34, thereby promoting the synthesis of a specific phosphoinositide, phosphatidylinositol-3-phosphate (PI3P), on endosomes [42]. Phosphoinositides are organelle-specific lipids and their phosphorylated head group binds to specific domains, such as the FYVE domain in EEA1, thereby allowing for the organelle-specific membrane association of proteins containing this domain 43, 44. Thus, the activation of Rab5 is a prerequisite to recruit specific proteins and promote the synthesis of specific lipids on endosomes, thereby determining the identity of the endosome, dictated by the proteins and lipids presented on the surface of the organelle.
Endosomal Maturation and Rab7 Activation While the endosome is remodeled, following further activation rounds of Rab5, additional fusion events enlarge the membrane content [45]. At a distinct point during this process, Rab5-GTP promotes the recruitment of the Rab7 GEF, the Mon1-Ccz1 complex [5] (Figure 2C). The PI3P content of the membrane also promotes this process, since Mon1-Ccz1 binds this lipid 46, 47. The Mon1-Ccz1 complex was initially identified in yeast and later in human cells 48, 49, 50. In both yeast and human cells, the complex is a specific GEF and triggers GTP loading of Rab7 51, 52. Subsequent analyses confirmed that the Mon1-Ccz1 complex is also the general Rab7 GEF in other organisms 53, 54, 55, 56, and uncovered the mechanism of nucleotide exchange [57]. Recent work suggested that there is a third, potentially regulatory, subunit of the Mon1-Ccz1 complex in mammals, named RMC1 [58], although in vitro studies 51, 52 and a recently published crystal structure [57] suggest that the two core subunits are sufficient for GEF-activity. Mon1-Ccz1 and Rab7 are required not only along the endosomal pathway, but also for autophagy 49, 59, 60. Rab7 has been identified on autophagosomes in flies, which require Mon1-Ccz1 [61]. Recently, it was shown that yeast Mon1-Ccz1 requires the LC3-like Atg8 protein, an early marker of growing autophagosomes, to bind to this organelle [62]. Mon1-Ccz1 then recruits and activates Ypt7. This enables autophagosomes to tether and finally fuse with the yeast vacuole or metazoan lysosome 60, 63, 64, 65. Similarly, Mon1-Ccz1 has been found on damaged mitochondria in mammalian cells, where it is required in a Rab5-to-Rab7 cascade, and autophagosome formation [66], indicating that the same protein-machinery can be recruited to different organelles in order to finally allow fusion with the lysosome.