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.
Highlights
Membrane trafficking in eukaryotic cells is tightly controlled by a range of proteins, including markers of membrane identity and multisubunit tethering complexes
It is known that VPS33A must interact with VPS16 in order to be recruited to homotypic fusion and vacuole protein sorting (HOPS) and to localize correctly to target membranes [8,11,21]
The majority of proteins identified in our assay co-localized exclusively with either VPS33A or VPS33B, indicating that these proteins are found on different subcellular membranes
Summary
Membrane trafficking in eukaryotic cells is tightly controlled by a range of proteins, including markers of membrane identity (such as small GTPases) and multisubunit tethering complexes. VPS33B forms a complex with VIPAR ( known as SPE-39, VIPAS39 or VPS16B), and mutations in either of these proteins can cause arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome [14]. Arthrogryposis, renal dysfunction, and cholestasis syndrome is a multi-system disorder, with some symptoms attributable to the known functions of VPS33B and VIPAR in apical recycling pathways [14], post-Golgi collagen processing [13], and α-granule formation in megakaryocytes [17,18]. The differences in phenotype conferred by mutations in VPS33A and VPS33B confirm that these proteins act in distinct cellular pathways. A recent review suggested that VPS33B and VIPAR were members of a multi-subunit membrane tethering complex with an analogous organisation to CORVET and HOPS [20]. We identified known interactors of VPS33A and VPS33B, and novel VPS33B interactors, but did not find evidence for the existence of other members of a stable CHEVI complex beyond VPS33B and VIPAR
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