Cellubrevin and synaptobrevins: similar subcellular localization and biochemical properties in PC12 cells.

Abstract

Abstract. There is strong evidence to indicate that proteins of the synaptobrevin family play a key role in exocytosis. Synaptobrevin 1 and 2 are expressed at high concentration in brain where they are localized on synaptic vesicles. Cellubrevin, a very similar protein, has a widespread tissue distribution and in fibroblasts is localized on endosome-derived, transferrin receptor-positive vesicles. Since brain cellubrevin is not detectable in synaptic vesicles, we investigated whether cellubrevin and the synaptobrevins are differentially targeted when co-expressed in the same cell. We report that in the nervous system cellubrevin is expressed at significant levels only by glia and vascular cells. However, cellubrevin is coexpressed with the two synaptobrevins in PC12 cells, a neuroendocrine cell line which contains synaptic vesicle-like microvesicles. In PC12 cells, cellubrevin has a distribution very similar to that of synaptobrevin 1 and 2. The three proteins are targeted to neurites which exclude the transferrin receptor and are enriched in synaptic-like microvesicles and dense-core granules. They are recovered in the synaptic-like microvesicle peak of glycerol velocity gradients, have a similar distribution in isopycnic fractionation and are coprecipitated by anti-synaptobrevin 2 immunobeads. Finally, cellubrevin, like the synaptobrevins, interact with the neuronal t-SNAREs syntaxin 1 and SNAP-25. These results suggest that cellubrevin and the synaptobrevins have similar function and do not play a specialized role in constitutive and regulated exocytosis, respectively.

A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

“…As expected, Ca 2ϩ -dependent secretion of hGH was decreased (to 32% of control) by the presence of botB/LC in cells transfected with a plasmid containing wild-type syb2 ( Figure 5B). Such profound, but incomplete inhibition of release has been described previously (Chilcote et al, 1995;Quetglas et al, 2002). The expression of botB/LC-resistant syb2(Q76V,F77W) restored secretion (104% of control) in the presence of toxin ( Figure 5B).…”

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

“…VAMP3 binds to plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25 [22,29,30,34]. Using the cell fusion assay in the current study, we examined the membrane fusion capacity of the ternary complexes formed by VAMP3 and the plasma membrane t-SNAREs.…”

“…VAMP3 forms complexes with syntaxin1, syntaxin4, SNAP-23 and SNAP-25 [29][30][31]. It has been reported that VAMP3 forms ternary complexes with syntaxin4/SNAP-25 [22] and syntaxin4/SNAP-23 [34].…”

“…Biochemical studies show that VAMP3 binds to plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25 [29][30][31]. Syntaxin4 is a homologue of syntaxin1, whereas SNAP-23 is a homologue of SNAP-25 [32,33].…”

Cross Talk between Tetanus Neurotoxin-insensitive Vesicle-associated Membrane Protein-mediated Transport and L1-mediated Adhesion

“…Polyclonal rabbit antibodies against mouse L1 and Fab fragments generated from this antibody were described previously (Rathjen and Rutishauser, 1984). The polyclonal anti-Syb 2 antibody (MC23) was described previously (Chilcote et al, 1995). The following commercial antibodies were used in this study: monoclonal anti-Na/K-ATPase a-1 (clone C464.6) from Upstate Biotechnology (Charlottesville, VA), monoclonal antisyntaxin 13 (clone 15G2) from StressGen (San Diego, CA), and monoclonal anti-Vti1b (Transduction Laboratories clone 7) from BD Biosciences (Franklin Lakes, NJ).…”