Our main conclusions are that the CORVET subunits Vps3 and Vps8, and the HOPS subunits Vps39 and Vps41, form discrete complex-specific subassemblies that function in Rab recognition. The activities of these subassemblies are coordinated with the core through contacts between the CTDs of Vps3 or Vps39 and the CTD of Vps11. The core subunits Vps16 and Vps33 also form a stable subassembly. Vps33 belongs to the Sec1-Munc18 (SM) family of cofactors (Sudhof and Rothman, 2009
), which are universally required for SNARE-mediated membrane fusion, so Vps16 and Vps33 likely link the Rab signaling activities of HOPS and CORVET to SNARE-mediated membrane fusion.
In early studies, HOPS was identified as an effector of Ypt7-GTP (Price et al.
; Seals et al.
) and the Vps39 subunit was reported to be a Ypt7 GEF (Wurmser et al.
; Binda et al.
). Vps41 binds Ypt7-GTP directly (), which explains why Vps41 is essential for stable binding of the HOPS holocomplex to Ypt7-GTP (Brett et al.
). However, although Vps39 shows a lack of nucleotide specificity in Ypt7 binding, as might be expected for a GEF, highly purified preparations of Vps39 or Vps39–860Δ failed to exhibit any nucleotide exchange activity in fluorescence and radioisotopic assays that readily detected the activities of bona fide GEFs (Nordmann et al.
; Ostrowicz et al.
; Lobingier, Brett, and Merz, unpublished results). Instead, the major Ypt7 GEF activity appears to reside within the Ccz1/Mon1 complex (Kucharczyk et al.
; Nordmann et al.
). The Vps39 CTD is dispensable for Vps39 binding to Ypt7 and Vps41 ( and ; Supplemental Table S2), and for binding of HOPS to Ypt7-GTPγS (Brett and Merz, unpublished results). The Vps39 CTD is, however, essential for Vps39 function in vivo, because its deletion phenocopies a vps39
Δ null mutant ( and Supplemental Table S2; Wurmser et al.
Like Vps39 and Vps41, the CORVET subunits Vps3 and Vps8 function as a Rab-recognition module that signals through Vps11. As with Vps39, the Vps3 CTD binds the Vps11 CTD, and this domain is needed for Vps3 function. In Y2H analyses we identified Vps21-interacting domains in both Vps3 and Vps8. While this manuscript was in preparation, Y2H hits between Vps8 and Vps21 were reported by two other groups (Markgraf et al.
; Pawelec et al.
). Moreover, we obtained genetic data, using the VPS8–200
suppressor of vps11–445
Δ, that Vps8 and Vps21 functionally interact. In our Y2H analyses, both Vps8 and Vps8–200 exhibited a preference for Vps21-GTP. In our experiments, Vps3 always exhibited preferential binding to activated Vps21. Native Vps3, as a CORVET substituent, is selectively retained on Vps21-GTP (). Using purified proteins we demonstrated that Vps3 binds Vps21-GTPγS directly (). In contrast, recent work suggested that Vps3 contributes to GDP-selective Vps21 binding by the CORVET holocomplex, and it was proposed in this work that Vps3 might function as a Vps21 GEF (Peplowska et al.
). However, as with Vps39, we have been unable to detect any nucleotide exchange activity mediated by purified Vps3 using assays that readily detect exchange mediated by a bona fide Vps21 GEF, Vps9 (Lobingier, Brett, and Merz, unpublished results). While this article was under revision, similar results for Vps3 were reported by Ostrowicz et al.
The distinct nucleotide specificities of the Rab-recognition modules within CORVET and HOPS are particularly striking. The CORVET subunits Vps3 and Vps8 appear to simply search for the presence or absence of Vps21-GTP. In contrast, the HOPS Rab-recognition module contains one subunit, Vps41, that detects Ypt7-GTP and a second subunit, Vps39, that is insensitive to the nucleotide binding state of Ypt7. HOPS might therefore monitor not only the presence of Ypt7-GTP but also the ratio of Ypt7-GTP to Ypt7 GDP. The functional consequences of the divergent Rab-binding modalities of HOPS and CORVET are not yet understood.
As membrane traffic traverses successive endolysosomal organelles, the Rab5 orthologue Vps21 is replaced by the Rab7 orthologue Ypt7 (). CORVET and HOPS, operating in concert with the GEFs Vps9 and Ccz1-Mon1, appear to control this Rab cascade (Rink et al.
; Vonderheit and Helenius, 2005
; Peplowska et al.
). Vps11 appears to be a central scaffold upon which both HOPS and CORVET assemble. Deletion analyses of Vps11 ( and Supplemental Table S2) confirm the functional importance of the Vps11 CTD. The almost complete functional complementation in trans of two Vps11 truncation mutants (–735Δ and –926Δ) by a C-terminal (736–1029) fragment of Vps11—which contains a binding site or sites for the C-termini of Vps3 and Vps39—underscores the modularity of this region. Vps11 may be a simple structural scaffold or it could have a more dynamic role in linking Rab signaling to Vps-C outputs, including the SNARE machinery that mediates fusion at the endosome and vacuole. In either case, the Vps11 CTD is now the prime candidate for the location at which switching between the HOPS and CORVET Vps-C configurations might occur. Consistent with the idea that the Vps11 CTD is a key element in this cascade, the C-terminal RING domain of Vps11 appears to function predominantly in fusion at the vacuole ( and Supplemental Table S4). Moreover, we recently isolated new vps11
alleles bearing mutations solely in the Vps11 CTD and exhibiting selective defects in traffic through late endosomes, to the vacuole, or both (Nickerson, Fawcett, and Merz, unpublished results). We are now working to understand how Rab signaling through Vps-C complexes drives fusion and other “output” processes (Nickerson et al.
) and how Vps-C complexes signal in time and space to transport cargoes derived from early endosomes, the late Golgi, autophagosomes, and cytoplasm-to-vacuole transport (CVT) vesicles through the endolysosomal system.
Interactions of HOPS and CORVET subunits with one another and with cognate Rabs. The subunit interaction topology summarized in this diagram integrates both biochemical and Y2H data. The similar diagram in summarizes only Y2H interactions.