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.
Highlights
It has been recognized for many years that there are two pathways, constitutive and regulated, by which secretory proteins are released from cells
Expression of vesicular stomatitis virus glycoprotein (VSV-G) in COS-1 Cells Cotransfected with Syntaxin 1A—COS-1 cells were transfected with a plasmid which encodes a form of VSV-G which is constitutively inserted into the plasma membrane
While there is abundant evidence that syntaxin 1A is important for secretion [28, 30], the specific functions it serves in the regulated secretory pathway are not well understood
Summary
Plasmids—Human GH was expressed with pXGH5 which is under control of the mouse metallothionein I promoter [41]. In some experiments (noted in legends to figures), the glycerol shock step was omitted, and the DNA precipitate was left on the cells for 24 h before addition of fresh culture medium containing cytosine arabinofuranoside. Cells were incubated with the primary antibody to VSV-G at 4 °C for 2 h and rinsed before fixation and permeabilization The following primary antibodies were used: hGH, rabbit anti-human pituitary GH polyclonal antibody (1: 1000, National Hormone and Pituitary Program, NIDDKD) or mouse anti-hGH (Zymed, San Francisco, CA); syntaxin 1A and syn 1A⌬M, monoclonal antibody to syntaxin 1A [44] (HPC-1, 1:1000); VSV-G, rabbit polyclonal antibody Materials—Reagents were received from the following sources: 125Ilabeled goat anti-rabbit secondary antibody (ICN); cell culture reagents, including trypsin-versene, gentamicin, penicillin/streptomycin, glutamine, and DMEM (BioWhittaker). All other reagents, including fetal bovine serum, were obtained from Sigma
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