The role of palmitoylation in trafficking of the t-SNAREs, SNAP-25 and syntaxin 11

SNARES (s&barbelow;oluble N&barbelow;-ethylmaleimide-sensitive factor a&barbelow;ttachment protein receptors) are a family of proteins that play an essential role in vesicle trafficking. Fusion of vesicles with target membranes is mediated by the formation of complexes between v-SNAREs localized on the vesicle membrane and t-SNAREs on the target membrane. Each membrane compartment is associated with a unique set of SNAREs. However, the mechanisms by which SNAREs are sorted to their resident membranes are poorly understood. The goal of this work was to understand the role of palmitoylation in membrane trafficking of two palmitoylated t-SNAREs, SNAP-25 and syntaxin 11.; It has been proposed that interactions of SNAP-25 with syntaxin 1A are required for initial membrane attachment of SNAP-25 (Vogel et al. J. Biol. Chem. 2000). However, residues 85–120 of SNAP-25, which do not interact with syntaxin 1A, were shown to be necessary and sufficient for plasma membrane targeting (Gonzalo et al. J. Biol. Chem. 1999). To differentiate between these two models of SNAP-25 membrane interactions, I studied a SNAP-25 point mutant (G43D) that is unable to interact with syntaxin 1A. SNAP-25 G43D has the same kinetics of membrane association as wildtype. Furthermore, SNAP-25 G43D is palmitoylated and localizes to the plasma membrane. Exogenously expressed SNAP-25 targets efficiently to the plasma membrane in cells of neuronal origin, but only partially in HeLa cells, a neurosecretion incompetent line. I hypothesize the existence of a neuronal specific factor, other than syntaxin 1A, that is required for SNAP-25 plasma membrane localization.; Recently, an atypical SNARE has been identified that has a C-terminal cysteine rich domain in lieu of the more common transmembrane domain. I sought to establish that syntaxin 11 is palmitoylated, and to determine a role for palmitoylation in its membrane attachment and localization. Syntaxin 11 is modified by palmitate, and palmitoylation is dependent on the cysteines within the C-terminal domain. Palmitoylation is not essential for syntaxin 11 membrane attachment or localization on intracellular membranes in non-polarized cells. However, disruption of palmitoylation either by site directed mutagenesis of the cysteine rich region or by treatment with 2-bromopalmitate, inhibits the localization of syntaxin 11 at the plasma membrane in polarized MDCK cells. Thus, palmitoylation is critical for the redistribution of syntaxin 11 to the plasma membrane from intracellular membranes in polarized cells.