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. 1998 Jun;9(6):1437-48.
doi: 10.1091/mbc.9.6.1437.

A novel tetanus neurotoxin-insensitive vesicle-associated membrane protein in SNARE complexes of the apical plasma membrane of epithelial cells

T Galli  1 , A Zahraoui, V V Vaidyanathan, G Raposo, J M Tian, M Karin, H Niemann, D Louvard
Affiliations
Free PMC article

A novel tetanus neurotoxin-insensitive vesicle-associated membrane protein in SNARE complexes of the apical plasma membrane of epithelial cells

T Galli et al. Mol Biol Cell. 1998 Jun.
Free PMC article

Abstract

The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP-containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.

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Figures

Figure 1
Figure 1
(A) TI-VAMP belongs to the VAMP/brevin family. Alignment of human TI-VAMP protein with the human brevins was made using Pileup software (GCG). The two potential coiled coils of TI-VAMP predicted by COILS (version 2.1) (Lupas et al., 1991) are underlined. Four hepta repeats of hydrophobic residues (marked by * under the corresponding amino acid in TI-VAMP) conserved within the VAMP/brevin family overlap with these domains. The domains involved in the binding of clostridial NTs are residues 38–47 (domain V1) and 62–71 (domain V2) in synaptobrevin 2 (Rossetto et al., 1994). Key amino acids (underlined below) are changed in the clostridial NT binding domains of TI-VAMP: 43VDIMR47 in domain V1 of synaptobrevin 2 → 137KGIMV141 in TI-VAMP and 62ELDDRADALQ71 in domain V2 of synaptobrevin 2 → 56LLIDKTENLV65 and in the cleavage sites. (B) A domain in the N-terminal portion of TI-VAMP (amino acids 80–119) shares similarity with human annexin XIII (GenBank accession number P27216) within its first annexin repeat. The alignment was performed using Bestfit (GCG). The similarity is 47.5%, and the identity is 27.5%. (C) Identification of TI-VAMP in CaCo-2 cell extract by TG11 rabbit serum. A CaCo-2 cell extract was immunoprecipitated with anti-human cellubrevin (ip hCB) as a control or with anti–TI-VAMP (ip TI-VAMP) (the pellet fractions represent a 10-fold enrichment compared with the amount in the extract). The bound fractions were run on SDS-PAGE (Schagger and von Jagow, 1987) and analyzed by Western blotting with TG11 rabbit serum. TI-VAMP appears as a 25-kDa band specifically immunoprecipitated by anti–TI-VAMP immunobeads.
Figure 2
Figure 2
Effects of recombinant light chain of clostridial NTs on SNARE proteins. Cellubrevin, TI-VAMP (A), SNAP25, SNAP23 (B), syntaxin 1a, and syntaxin 3 (C) were translated in vitro in the presence of [35S]methionine and then incubated with the various light chains of the NTs in 150 mM potassium glutamate, 10 mM HEPES-KOH, pH 7.2, for 12 h at 37°C. The cleavage was analyzed after the products were separated by SDS-PAGE and visualization of bands by autoradiography. The concentrations of the toxins used were as follows: TeNT, BoNT/B, D, F, and G, 2 μM; BoNT A, 0.8 μM; BoNT C, 0.9 μM; and BoNT E, 0.4 μM.
Figure 3
Figure 3
Localization of v- and t-SNARES in vertical (A and B) and horizontal (C–F) confocal sections of CaCo-2 cells. Syntaxin 3 (green, A) and SNAP23 (green, B) are present at the apical plasmalemma. The staining for E-cadherin, a protein of the basolateral membrane, is in red (A and B). Note the occurrence of a low amount of SNAP23 in the top part of the lateral plasma membrane (yellow, B). Cellubrevin (green, C, E) and TI-VAMP (green, D, F) are present on intracellular organelles localized both in the lateral plane (C and D) and in the apical domain (E and F). Note the very low degree of colocalization (yellow) with transferrin receptor (monoclonal antibody H68.4, red, C and D) of both v-SNAREs. Bar, 10 μm.
Figure 4
Figure 4
Localization of t-SNARES in horizontal confocal sections of CaCo-2 cells. Double immunofluorescence micrographs of CaCo-2 cells stained for SNAP23 and ZO-1, a tight junction marker (left) or syntaxin 3(Stx3) and E-cadherin, a basolateral marker (right). Syntaxin 3 is restricted to the apical plasma membrane (note the lack of staining of the lateral plasma membrane that is positive for E-cadherin in bottom right micrographs). SNAP23 is highly concentrated at the apical plasmalemma and is present in tight junctions (top left micrographs; arrow indicates colocalization with ZO-1) and at low level in the lateral plasma membrane below tight junctions (bottom left). Bar, 10 μm.
Figure 5
Figure 5
Immunogold localization of syntaxin 3, SNAP23, and TI-VAMP in CaCo-2 cells. Syntaxin 3 (A), SNAP23 (B), and TI-VAMP (C) visualized with their respective affinity-purified antibody and PAG 10 are localized at the cytoplasmic side of the microvillar membrane of polarized CaCo-2 cells. Bars, 200 nm.
Figure 6
Figure 6
In vitro formation of SNARE complexes of cellubrevin and TI-VAMP with SNAP23 and syntaxin 3. Glutathione beads coated with GST, GST-hCB1 (first coiled coil only), GST-hCB2 (whole cytoplasmic domain), or GST–TI-VAMP (whole cytoplasmic domain) (1 μM, 100 μl) were incubated overnight with radiolabeled in vitro–translated human syntaxin 3 (stx3) alone or together with human SNAP23 (8 μl of translated proteins). The fractions bound to glutathione beads were eluted, separated by SDS-PAGE, and visualized by autoradiography. The shorter forms of stx3 (stx3Δ1 and stx3Δ2) are likely to be generated by initiation of translation at internal methionine residues. Binding of syntaxin 3 alone to the v-SNAREs is at the limit of detection. The molecular weights of the SDS stable complexes are 85 and 120 kDa in the case of GST-hCB2 and 115 and 150 kDa in the case of GST–TI-VAMP. Note also that the GST-hCB1 SNARE complex is not SDS stable.
Figure 7
Figure 7
Effect of NEM on the formation of SNARE complexes in CaCo-2 cells. CaCo-2 cells grown to confluency were treated with 1 mM NEM for 15 min and then with 2 mM DTT for 15 min (NEM) or with 1 mM NEM plus 2 mM DTT for 30 min (NEM+DTT) on ice. The cells were then further incubated in culture medium for 30 min at 37°C and lysed in the presence of 1% Triton X-100 in conditions in which proteins are diluted enough so that the SNARE complex does not form spontaneously (Banerjee et al., 1996). The cell extract was immunoprecipitated with anti-syntaxin 3 (ip stx3) or anti-GST (ip gst) (the pellet fractions represent a fivefold enrichment compared with the amount in the extract). The bound fractions were run on SDS-PAGE (Schagger and von Jagow, 1987) and analyzed by Western blotting with specific SNARE (syntaxin 3, stx3; hCB, human cellubrevin) antibodies. Note that syntaxin 3 is entirely immunoprecipitated and that NEM treatment greatly stimulates the recovery of SNAP23, cellubrevin, and TI-VAMP in the syntaxin 3 immunobead pellets.
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