Evidence That Syntaxin 1A Is Involved in Storage in the Secretory Pathway

Abstract

Syntaxin 1A is a nervous system-specific protein thought to function during the late steps of the regulated secretory pathway by mediating the docking of secretory vesicles with the plasma membrane. We have examined the effects of transiently overexpressing syntaxin 1A on protein secretion in constitutively secreting cell lines that do not normally express the protein. Syntaxin 1A showed the constitutive release of marker proteins human growth hormone (hGH) and vesicular stomatitis virus glycoprotein from COS-1 cells, increasing the intracellular half-life of human growth hormone from 90 min to 18 h. A similar effect was observed in HEK 293 cells. Immunofluorescence microscopy revealed that these secretory proteins were concentrated in the periphery of the cell. The effect was specific for the full-length neuronal protein. Neither a syntaxin 1A variant which lacks a membrane attachment domain nor syntaxin 2 caused the cells to retain human growth hormone. The effect of syntaxin 1A was partially reversed by incubating the cells with botulinum type C1 neurotoxin, which specifically cleaves syntaxin 1A. Release of human growth hormone from syntaxin 1A-expressing cells was maintained during a blockade of protein synthesis, suggesting that the hormone was being released from a pool of stored vesicles which accumulated before the addition of cycloheximide. The existence of a post-Golgi storage compartment in syntaxin 1A-expressing cells was confirmed using brefeldin A to collapse the Golgi stacks in both HEK 293 and COS-1 cells. Brefeldin A rapidly blocked growth hormone release in control cultures while having no effect on release in cells expressing syntaxin 1A. Reducing the temperature to 19 degrees C, which inhibits transport from the trans-Golgi network, also inhibited hGH secretion from cells without syntaxin 1A but had little effect on hGH secretion from cells with syntaxin 1A. The present experiments indicate that syntaxin 1A enables the storage of vesicles which would otherwise be immediately released.

Botulinum neurotoxin light chains inhibit both Ca2+‐induced and GTP analogue‐induced catecholamine release from permeabilised adrenal chromaffin cells

“…1, GppNHp stimulates catecholamine release above basal levels in the absence of added Ca 2+ with 5 mM EGTA present. This Ca2+-independent secretion triggered by GppNHp, as in previous work [33,34], is smaller than that triggered by 10 BM Ca 2+ but was reproducibly observed. The release due to GppNHp was 2.5 _+ 0.2% of total cellular catecholamine above basal from 17 cell batches.…”

“…Various GTP analogues have multiple effects on secretion from permeabilised chromaffin cells [47] which includes stimulation of release from permeabilised cells [33][34][35] and exocytosis in patch-clamped cells [36] by GppNHp at very low free Ca 2+ concentrations. Like Ca2+-induced secretion, Ca2+-independent catecholamine release stimulated by GppNHp is partially MgATP-dependent, is substantially inhibited by prior treatment with botulinum neurotoxin D and is partially inhibited by neurotoxins C1 and E. These results suggest that both activators of exocytosis activate a common mechanism leading to exocytosis that requires the SNARE proteins, VAMP, syntaxin and SNAP-25 and would argue against the presence of a SNARE-independent pathway for exocytosis in adrenal chromaffin cells.…”

“…Chromaffin cells express all three neurotoxin substrates [17,18] which can be isolated in a complex [18] and Ca2+-dependent exocytosis in these cells is sensitive to neurotoxins that target VAMP (tetanus and botulinum B neurotoxins) [19][20][21][22][23][24] or SNAP-25 (botulinum A and E neurotoxins) [23,[25][26][27][28]. Insulin-secreting cells are also sensitive to these two classes of neurotoxins [29][30][31] and studies on these cells have suggested that VAMP is essential for Ca2+-induced secretion but not for insulin release activated by the non-hydrolysable GTP analogue, GTPTS, since this was insensitive to tetanus toxin and botulinum B neurotoxin [29].…”

“…These data on insulin-secreting cells, coupled with studies on constitutive vesicle traffic in epithelial cells in which basolateral but not apical exocytosis is inhibited by botulinum neurotoxins [32], appear to suggest that exocytosis could occur by either SNARE-dependent or SNARE-independent pathways. Exocytosis in chromaffin cells is triggered by Ca 2+ [15,16] but exocytosis can also be activated in a CaZ+-independent manner by GTP analogues in permeabilised chromaffin cells [33][34][35][36]. We have, therefore, used botulinum neurotoxins to investigate whether exocytosis activated by a non-hydrolysable GTP analogue in permeabilised chromaffin cells, occurs by a SNARE-dependent mechanism based on sensitivity to recombinant neurotoxin light chains.…”

Adrenal chromaffin cells contain functionally different SNAP‐25 monomers and SNAP‐25/syntaxin heterodimers

“…Catecholamine release from adrenal chromaffin cells, vasopressin release from neurohypophysial terminals and insulin release from pancreatic 13 cells is inhibited by botulinum neurotoxin A [14][15][16][17][18]. However, the variable cleavage of SNAP-25 in pancreatic [3 cells, the incomplete inhibition of vasopressin and catecholamine release and the preferential attack of ATP-dependent priming by botulinum neurotoxin A suggested that this toxin may have molecular targets of different sensitivities [14][15][16][17][18][19][20][21].…”