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

Abstract

The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/169185 doi: bioRxiv preprint first posted online 2 ABSTRACT Eukaryotic cells use conserved multisubunit membrane tethering complexes, including CORVET and HOPS, 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 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.

VPS41recessive mutation causes ataxia and dystonia with retinal dystrophy and mental retardation by inhibiting HOPS function and mTORC1 signaling

“…VPS41 S285P interacted with both VPS18 and VPS33A, but interaction of VPS41 R662* with the other HOPS subunits was strongly reduced ( Figure 3A and S5). Since interaction between VPS41 and VPS18 requires the presence of the C-terminal RING domains that is absent from VPS41 R662* ( Figure S1), these data are in agreement with present literature (42).…”

A proteomic analysis unravels novel CORVET and HOPS proteins involved inToxoplasma gondiisecretory organelles biogenesis

“…Consistent with this, it has been shown that Vps8 binds through both the RING domain and a disordered C-terminus to both Vps18 and Vps11 subunits (Hunter et al, 2017). Our hypothesis is that TgVps8, in addition to acting as a sorting factor for ROP/MIC proteins, delivers proteases (such as ASP3), which are involved in the processing of pro-proteins, to secretory organelles.…”

“…Furthermore, we demonstrated that the endosomal accumulation of other subunits that are HOPS specific (Vps39), common to CORVET and HOPS (Vps18 and Vps11), or interacting with HOPS (CHC and CK3L) is severely compromised in Tgvps8 mutants. Consistent with this, it has been shown that Vps8 binds through both the RING domain and a disordered C‐terminus to both Vps18 and Vps11 subunits (Hunter et al, ). Our hypothesis is that TgVps8, in addition to acting as a sorting factor for ROP/MIC proteins, delivers proteases (such as ASP3), which are involved in the processing of pro‐proteins, to secretory organelles.…”

“…Interestingly, the results revealed >6 unique peptides that match the amino acid sequence of Vps9 domain‐containing protein (Vps9‐DCP, a putative guanine exchange factor known to interact with Rab5) and also >10 unique peptides that match a protein (TGME49_289520) with low similarity to the CORVET‐specific subunit Vps8. TGME49_289520 is a large protein of 3307 amino acids and possesses only conservation with the C‐terminal RING domain, which, in yeast, functions as a E3 ubiquitin ligase and is believed to regulate the fusion events at the vacuole, most likely through binding interactions with other fusion factors (Figure S2a; Budhidarmo, Nakatani, & Day, ; Hunter, Scourfield, Emmott, & Graham, ; Plemel et al, ). The presence of this Vps8‐like protein suggests that the CORVET complex is present in Toxoplasma .…”

“…Tetrahymena expresses only a CORVET complex, and surprisingly, Vps8a binds to Rab7. RILP: Rabinteracting lysosomal protein; TGFBRAP1: transforming growth factor-β receptor-associated protein 1 ubiquitin ligase and is believed to regulate the fusion events at the vacuole, most likely through binding interactions with other fusion factors ( Figure S2a; Budhidarmo, Nakatani, & Day, 2012;Hunter, Scourfield, Emmott, & Graham, 2017;Plemel et al, 2011). The presence of this Vps8-like protein suggests that the CORVET complex is present in Toxoplasma.…”