EVIDENCE THAT SYNTAXIN 1A IS INVOLVED IN STORAGE IN THE SECRETORY PATHWAY

Syntaxin 1A is a nervous system-specific protein thought to function d uring the late steps of the regulated secretory pathway by mediating t he docking of secretory vesicles with the plasma membrane. We have exa mined 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 m arker proteins human growth hormone (hGH) and vesicular stomatitis vir us glycoprotein from COS-1 cells, increasing the intracellular half-li fe of human growth hormone from 90 min to 18 h. A similar effect was o bserved in HEK 293 cells. Immunofluorescence microscopy revealed that these secretory proteins were concentrated in the periphery of the cel l. The effect was specific for the full-length neuronal protein. Neith er 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 bo tulinum type C-1 neurotoxin, which specifically cleaves syntaxin Ik Re lease of human growth hormone from syntaxin 1A-expressing cells was ma intained during a blockade of protein synthesis, suggesting that the h ormone was being released from a pool of stored vesicles which accumul ated before the addition of cycloheximide. The existence of a post-Gol gi storage compartment in syntaxin 1A-expressing cells was confirmed u sing 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 syntax in 1A. Reducing the temperature to 19 degrees C, which inhibits transp ort from the trans-Golgi network, also inhibited hGH secretion from ce lls without syntaxin 1A but had little effect on hGH secretion hom cel ls with syntaxin 1A. The present experiments indicate that syntaxin 1A enables the storage of vesicles which would otherwise be immediately released.