Abstract
Eukaryotic cells use conserved multisubunit membrane tethering complexes, including CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting), to control the fusion of endomembranes. These complexes have been extensively studied in yeast, but to date there have been far fewer studies of metazoan CORVET and HOPS. Both of these complexes comprise six subunits: a common four-subunit core and two unique subunits. Once assembled, these complexes function to recognise specific endosomal membrane markers and facilitate SNARE-mediated membrane fusion. CORVET promotes the homotypic fusion of early endosomes, while HOPS promotes the fusion of lysosomes to late endosomes and autophagosomes. Many of the subunits of both CORVET and HOPS contain putative C-terminal zinc-finger domains. Here, the contribution of these domains to the assembly of the human CORVET and HOPS complexes has been examined. Using biochemical techniques, we demonstrate that the zinc-containing RING (really interesting new gene) domains of human VPS18 and VPS41 interact directly to form a stable heterodimer. In cells, these RING domains are able to integrate into endogenous HOPS, showing that the VPS18 RING domain is required to recruit VPS41 to the core complex subunits. Importantly, this mechanism is not conserved throughout eukaryotes, as yeast Vps41 does not contain a C-terminal zinc-finger motif. The subunit analogous to VPS41 in human CORVET is VPS8, in which the RING domain has an additional C-terminal segment that is predicted to be disordered. Both the RING and disordered C-terminal domains are required for integration of VPS8 into endogenous CORVET complexes, suggesting that HOPS and CORVET recruit VPS41 and VPS8 via distinct molecular interactions.
Highlights
Eukaryotic cells exert tight control over the fusion of their membrane-bound compartments
We showed using purified recombinant proteins that the really interesting new gene (RING) domains of VPS18 and VPS41 form a stable, direct heterodimer (Figure 3) and that this interaction mediates recruitment of VPS41 to the homotypic fusion and vacuole protein sorting (HOPS) complex in mammalian cells (Figure 4)
Our finding that the VPS18 and VPS41 RING domains mediate HOPS assembly is in broad agreement with a previous study in which co-transfected, epitope-tagged HOPS subunits were co-immunoprecipitated by truncated forms of VPS18 and VPS41 [11]
Summary
Eukaryotic cells exert tight control over the fusion of their membrane-bound compartments. HOPS is required for the fusion of autophagosomes to lysosomes [6,7,8,9] In mammalian cells, these complexes share four core subunits (VPS11, VPS16, VPS33A and VPS18, collectively known as the class C core), along with two subunits unique for CORVET (VPS8 and TRAP-1) or HOPS (VPS39 and VPS41) (Figure 1) [5,10,11]. These complexes share four core subunits (VPS11, VPS16, VPS33A and VPS18, collectively known as the class C core), along with two subunits unique for CORVET (VPS8 and TRAP-1) or HOPS (VPS39 and VPS41) (Figure 1) [5,10,11] These non-core subunits direct the complexes to their different membrane targets: Rab5-positive endosomes for CORVET [5,12] and Rab7-positive for HOPS [13,14]
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