Proteomic and Biochemical Comparison of the Cellular Interaction Partners of Human VPS33A and VPS33B.
Journal of molecular biology
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Hunter, M. R., Hesketh, G. G., Benedyk, T., Gingras, A., & Graham, S. (2018). Proteomic and Biochemical Comparison of the Cellular Interaction Partners of Human VPS33A and VPS33B.. Journal of molecular biology, 430 (14), 2153-2163. https://doi.org/10.1016/j.jmb.2018.05.019
Multi-subunit tethering complexes control membrane fusion events in eukaryotic cells. CORVET and 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 utilised 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 these proteins have distinct sub-cellular localisations. Consistent with previous reports, we observed that VPS33A was co-localised with many components of class III phosphatidylinositol 3-kinase (PI3KC3) complexes: PIK3C3, PIK3R4, NRBF2, UVRAG and RUBICON. Although VPS33A clearly co-localised with several subunits of CORVET and HOPS in this assay, no proteins with the canonical CORVET/HOPS domain architecture were found to co-localise 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.
Cell Line, Humans, Proteins, Vesicular Transport Proteins, Protein Interaction Mapping, Proteomics, Protein Conformation, Models, Molecular, HEK293 Cells, Phosphatidylinositol 3-Kinase, THP-1 Cells
This work was supported by a Sir Henry Dale Fellowship, jointly funded by the Royal Society and the Wellcome Trust, to SCG [098406/Z/12/Z and 098406/Z/12/B] and an Isaac Newton Trust/Wellcome Trust ISSF/University of Cambridge Joint Research Grant to SCG. GGH was supported by a Parkinson Canada Basic Research Fellowship. ACG is a Tier 1 Canada Research Chair and supported by a CIHR Foundation grant (FDN 143301).
Wellcome Trust (098406/Z/12/Z)
Wellcome Trust (098406/Z/12/B)
External DOI: https://doi.org/10.1016/j.jmb.2018.05.019
This record's URL: https://www.repository.cam.ac.uk/handle/1810/279458
Attribution 4.0 International (CC BY 4.0)
Licence URL: https://creativecommons.org/licenses/by/4.0/