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

doi:10.1074/jbc.271.19.11214

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

From the (1)Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan 48109 and the
(2)Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

^§ To whom correspondence should be addressed:
M 1301 MSRB III, Dept. of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109-0632.
Tel.: 313-747-2257; Fax: 313-763-4450; mbittner{at}umich.edu.

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 slowed 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 C 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°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.

Footnotes

↵* This study was supported in part by National Science Foundation Grant IBN 9008685 (to M. A. B.) and United States Public Health Service Grant R01 DK27959 (to R. W. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

TGN

trans-Golgi network

DMEM

Dulbecco's modified Eagle's medium

HA

hemagglutinin

HEK

human embryonic kidney

hGH

human growth hormone

NSF

N-ethylmaleimide-sensitive fusion protein

α-SNAP

soluble NSF attachment protein

SNAP-25

synaptosome-associated protein of 25 kDa

t-SNARE

target SNAP receptor

v-SNARE

vesicular SNAP receptor

VAMP-2

vesicle-associated membrane protein or synaptobrevin-2

VSV-G

vesicular stomatitis virus glycoprotein

BFA

brefeldin A.
↵²M. A. Bittner and R. W. Holz, unpublished observations.
↵³When culture medium was changed at the beginning of the incubation with drug, we observed a brief burst of hGH secretion from syntaxin 1A-expressing cells, apparently as a result of mechanical stimulation (see also Fig. 7). While the significance of this observation is not yet clear, it is consistent with the presence of a pool of stored hGH which is available for release.
↵⁴During a 3-h incubation, cells without BFA secreted 3.9 ng of hGH/well, compared with 0.6 ng/well for cells treated with BFA (Fig. 6A). One might have expected the difference (3.3 ng/well) to accumulate in the BFA-treated cells, yet the actual increase of 0.6 ng/well (-BFA, 0.98 ± 0.05; +BFA, 1.61 ± 0.09) was much less. Interference with vesicular trafficking over this prolonged period may have affected the expression of the protein.
- Received July 13, 1995.
- Revision received February 16, 1996.