Membrane localization and biological activity of SNAP-25 cysteine mutants in insulin-secreting cells

The tSNARE SNAP-25 is expressed in pancreatic beta-cells and is involved in the regulated release of insulin. It has been shown previously that SNAP-2 5 associates with the plasma membrane consequent to palmitoylation of one o r more cysteines in the central region of the molecule. The importance of p almitolyation in the biological function of SNAP-25 in exocytosis was not a ddressed. Furthermore, studies on both SNAP-25 and its non-palmitoylated ho mologues SNAP-29 and sec9, have suggested an alternative or complementary m echanism for membrane association involving interaction with syntaxin, To a ddress these issues, we have now studied the behavior and biological activi ty of cysteine mutant SNAP-25 in insulin-secreting (HIT) cells. While 91% o f native SNAP-25 was associated with the membrane, this value decreased to 56% for the single cysteine mutant C85/A and to 10% for the double (C85,88/ A) and quadruple (C85,88,90,92/A) mutants, The mutant SNAP-25 forms were al l found to bind syntaxin 1A with equal efficacy. Over-expression of syntaxi n 1A in HIT cells allowed for partial relocalization of both the double and quadruple SNAP-25 cys mutants to the membrane. By introducing a further mu tation to the SNAP-25 molecules to render them resistant to botulinum neuro toxin E, it was possible to study their ability to reconstitute regulated i nsulin secretion in toxin-treated HIT cells. Native SNAP-25 was able to ful ly reconstitute secretory activity in such cells. Despite the fact that the single cysteine mutant was significantly displaced to the cytosol, it stil l displayed 82% activity in the secretion reconstitution assay, and a simil ar discrepancy was seen for the double mutant. Even the quadruple mutant wi th no remaining cysteines was able to support a minimal level of secretion, It is concluded that both palmitoylation and binding to syntaxin are impli cated in membrane association of SNAP-25, This as well as the discrepancy b etween membrane localization and biological activity of the cysteine mutant s, suggests a complex, multi-component process for association of SNAP-25 w ith the membrane and its recruitment to a biologically productive state.