The CORVET Subunit Vps8 Cooperates with the Rab5 Homolog Vps21 to Induce Clustering of Late Endosomal Compartments

Abstract

Membrane tethering, the process of mediating the first contact between membranes destined for fusion, requires specialized multisubunit protein complexes and Rab GTPases. In the yeast endolysosomal system, the hexameric HOPS tethering complex cooperates with the Rab7 homolog Ypt7 to promote homotypic fusion at the vacuole, whereas the recently identified homologous CORVET complex acts at the level of late endosomes. Here, we have further functionally characterized the CORVET-specific subunit Vps8 and its relationship to the remaining subunits using an in vivo approach that allows the monitoring of late endosome biogenesis. In particular, our results indicate that Vps8 interacts and cooperates with the activated Rab5 homolog Vps21 to induce the clustering of late endosomal membranes, indicating that Vps8 is the effector subunit of the CORVET complex. This clustering, however, requires Vps3, Vps16, and Vps33 but not the remaining CORVET subunits. These data thus suggest that the CORVET complex is built of subunits with distinct activities and potentially, their sequential assembly could regulate tethering and successive fusion at the late endosomes.

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

“…Both CORVET and Vac1 have been described as Vps21 effectors (12,14,16,17,23,24). In agreement with this, all three GFP-tagged proteins colocalized with Vps21 (Fig.…”

“…Electron microscopy of Epon-embedded cells was done as described previously (14,31,32). Statistical evaluation of the EM sections was performed by 50 randomly selected cell profiles and counting of the number of vesicles and putative endosomal structures.…”

“…Previous data provided support that five factors, the Rab5 GTPase Vps21, its GEF Vps9, the Q-SNARE Pep12, the Sec1/Munc18-like Vps45 protein, and EEA1-like Vac1 protein cooperate at the early endosome (23,24). With the identification of the CORVET complex, it was assumed that COR-VET acts independently of the Vac1-mediated tethering at early endosomes (12,14), although the possible interdependence was not addressed in any previous study. We decided to address this issue by comparing the different mutants and following the localization of both tethers.…”

“…On the basis of its homology to HOPS, the CORVET complex has been postulated to act as a tethering complex as well (2,4,12,14). However, it has remained challenging to find solid evidence supporting such a function.…”

“…This tethering machinery interacts with the SNARE Pep12 and its Sec1/Munc18 protein Vps45 to fuse transport vesicles at the endosomes (9,11,13,24). Additional fusion events at the early and late endosome may require the CORVET complex (12,14) (Fig. 1A).…”