VPS18 recruits VPS41 to the human HOPS complex via a RING–RING interaction

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.

CORVET, CHEVI and HOPS – multisubunit tethers of the endo-lysosomal system in health and disease

“…Vps11, Vps8, Vps41 and Vps18: potential E3 ubiquitin ligases Vps11, Vps8, Vps41 and Vps18 all have 'really interesting new gene' (RING) finger domains (Horazdovsky et al, 1996;Radisky et al, 1997;Rieder and Emr, 1997). Vps18 and Vps41 interact with each other via these RING domains, and this is required for formation of the HOPS complex (Hunter et al, 2017). RING domains typically confer E3 ubiquitin ligase activity to a protein, allowing them to recruit ubiquitin-conjugating enzymes and transfer ubiquitin to a specific substrate (Deshaies and Joazeiro, 2009).…”

Proteomic and Biochemical Comparison of the Cellular Interaction Partners of Human VPS33A and VPS33B

“…F3290). GFP and Myc immunoprecipitations were performed using GFP-Trap and Myc-Trap resin (ChromoTek), respectively, as in [61] . GST pull-down experiments were performed as described in [61] , using 40–100 pmol purified VPS33B/GST-VIPAR complex as bait (described above).…”

“…All samples were immunoblotted as per [61] . Immunoblot band intensity was quantified using Image Studio Lite (version 5.4, LI-COR) and non-linear fitting to a Gaussian distribution was performed using Prism 7 (GraphPad).…”

Non-native fold of the putative VPS39 zinc finger domain

“…Previous studies using truncation mapping have highlighted the importance of the C-terminal regions of HOPS components in assembly of the HOPS complex 3 , 14 – 16 . Recruitment of VPS41 to the class C core is facilitated by the C-terminal RING (really interesting new gene) domains of VPS18 and VPS41, which interact directly 15 . RING domains are a type of zinc finger, with an eight-residue motif containing six or seven cysteine residues and one or two histidine residues that coordinate two zinc ions 17 – 19 .…”

“…The C terminus of VPS39 contains a putative zinc finger domain 15 ( Figure 1A ), the closest homologue of which is the zinc finger domain of Saccharomyces cerevisiae protein Pcf11 ( Figure 1B, C ) 20 . This putative VPS39 zinc finger domain is much shorter than those of VPS18 and VPS41, and is predicted to bind only one zinc ion via four ligands 15 .…”