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

Abstract

Multi-subunit tethering complexes control membrane fusion events in eukaryotic cells. Class C core vacuole/endosome tethering (CORVET) and homotypic fusion and vacuole protein sorting (HOPS) are two such complexes, both containing the Sec1/Munc18 protein subunit VPS33A. Metazoans additionally possess VPS33B, which has considerable sequence similarity to VPS33A but does not integrate into CORVET or HOPS complexes and instead stably interacts with VIPAR. It has been recently suggested that VPS33B and VIPAR comprise two subunits of a novel multi-subunit tethering complex (named “CHEVI”), perhaps analogous in configuration to CORVET and HOPS. We utilized the BioID proximity biotinylation assay to compare and contrast the interactomes of VPS33A and VPS33B. Overall, few proteins were identified as associating with both VPS33A and VPS33B, suggesting that these proteins have distinct sub-cellular localizations. Consistent with previous reports, we observed that VPS33A was co-localized with many components of class III phosphatidylinositol 3-kinase (PI3KC3) complexes: PIK3C3, PIK3R4, NRBF2, UVRAG and RUBICON. Although VPS33A clearly co-localized with several subunits of CORVET and HOPS in this assay, no proteins with the canonical CORVET/HOPS domain architecture were found to co-localize with VPS33B. Instead, we identified that VPS33B interacts directly with CCDC22, a member of the CCC complex. CCDC22 does not co-fractionate with VPS33B and VIPAR in gel filtration of human cell lysates, suggesting that CCDC22 interacts transiently with VPS33B/VIPAR rather than forming a stable complex with these proteins in cells. We also observed that the protein complex containing VPS33B and VIPAR is considerably smaller than CORVET/HOPS, suggesting that the CHEVI complex comprises just VPS33B and VIPAR.

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

“…Vps33B is a homolog of Vps33A, whereas VIPAS39 shares significant similarity to the C-terminal region of Vps16 (Gissen et al, 2005;Graham et al, 2013). Despite these homologies neither VIPAS39 nor Vps33B are incorporated into HOPS or CORVET (Cullinane et al, 2010;Hunter et al, 2018;Rogerson and Gissen, 2016;Van Der Kant et al, 2015). Recruitment of Vps33A to the human HOPS complex requires the Vps16 residues 642-736 (Graham et al, 2013).…”

“…As discussed in the context of the Vps3-Vps8 recycling pathway, the CCC and Retriever complexes are involved in the sorting of cargoes bearing an NPxY/NxxY motif, including integrins, into a specialized endosomal recycling pathway (McNally et al, 2017). Recent studies have shown that Vps33B interacts with CCDC22 of the CCC complex (Hunter et al, 2018), binds integrin β subunits (Xiang et al, 2015) and is required for integrin recycling (Rogerson and Gissen, 2018). This functionally links Vps33B to the CCC and Retriever complexes and raises the question of whether Vps33B can recruit the CCC complex to Rab11-positive recycling endosomes for the formation of recycling tubules.…”

“…Mammalian Vps41 also interacts with AP-3 (Asensio et al, 2013). dependent integrin recycling pathways has not yet been investigated (Hunter et al, 2018;Jonker et al, 2018;Van Der Kant et al, 2015). There are no indications for molecular interactions between Vps3 or Vps8 and CHEVI (Hunter et al, 2018).…”

“…Likewise, depletion of VIPAS39 leads to an increase in LAMP-1-positive compartments, that is late endosomes and lysosomes (Cullinane et al, 2010). Since neither Vps33B nor VIPAS39 are incorporated into HOPS, this role is complementary to the HOPS-dependent late endosome-lysosome fusion (Graham et al, 2013;Hunter et al, 2018;Van Der Kant et al, 2015). In addition, CHEVI has been implicated in phagosome maturation.…”

Diversification of CORVET tethers facilitates transport complexity inTetrahymena thermophila

“…CHEVI is associated with both conserved, lineage-and tissue-specific structures, e.g., α-granules and lamellar bodies, which are mammalian platelet-specific and keratinocyte-specific lysosome-related organelles (LROs) (Bem et al, 2015;Dai et al, 2016;Lo et al, 2005;Rogerson and Gissen, 2018). A similar configuration pertains for Drosophila Vps16B and Vps33B, which also form a novel complex (Cullinane et al, 2010;Gissen et al, 2004;Hunter et al, 2018;Pulipparacharuvil et al, 2005;Tornieri et al, 2013). Further, HOPS or CORVET subunits may function in stable sub-complexes.…”