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《Cell reports》2023,42(8):112852
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Proteomic and Biochemical Comparison of the Cellular Interaction Partners of Human VPS33A and VPS33B
Morag R. Hunter Geoffrey G. Hesketh Tomasz H. Benedyk Anne-Claude Gingras Stephen C. Graham 《Journal of molecular biology》2018,430(14):2153-2163
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. 相似文献
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1. The possibility that a long-lasting neuronal activation regulates the expression of muscarinic cholinergic receptors was studied with three cultured neuronal cell lines. 2. Continuous depolarization of a subclone of the neuroblastoma-glioma NG108-15 hybrid cells with potassium chloride increased by 45-75% the number of cholinergic muscarinic receptors, monitored with 3H-QNB, whereas a short incubation with KCl for 10 min or 6 hr had no effect. 3. The calcium channel blocker verapamil increased the effect of KCl. 4. Two cell lines, named SC9 and WC5, that originate from the rat brain, also bind 3H-QNB. They were therefore used to test whether the effect of chronic depolarization is universal. Depolarized SC9 and WC5 cells, in the presence or absence of verapamil, did not show an increased 3H-QNB binding. 5. Muscarinic receptors of both SC9 and WC5 cells have a higher affinity to pirenzepine than the M-3 receptor subtype of the neuroblastoma-glioma cells, suggesting therefore that the two rat brain cell lines possess M-1 or M-2 receptors. 6. The physiological significance of this differential role of depolarization on the expression of different muscarinic receptors is discussed in the context of their postreceptor second messengers. 相似文献