Syntaxin-1A is excluded from recycling synaptic vesicles at nerve terminals

At presynaptic terminals, intermixing during cycles of exocytosis and endocytosis challenges the molecular identity of the plasma and synaptic vesicle membranes. Although synaptic vesicle components are retrieved during recycling, the extent to which plasma membrane proteins enter the synaptic vesicle recycling pathway has not been examined. The target-SNARE (N-ethylmaleimide- sensitive factor attachment protein receptor) syntaxin-1 was shown previously to be present on putative synaptic vesicular membranes (Koh et al., 1993; Walch-Solimena et al., 1995; Kretzschmar et al., 1996), suggesting that syntaxin may cycle between the synaptic vesicle pool and the cell surface (Walch-Solimena et al., 1995). This implies that the molecular identity of the two membranes is not maintained during synaptic activity. Because the main role of syntaxin-1 is as a target-SNARE for vesicle fusion, appearance on synaptic vesicles could lead to futile interactions with vesicle- SNARE proteins. We investigated whether the subcellular localization of syntaxin- 1A, tagged with the pH-sensitive fluorescent tag pHluorin, is regulated during neurotransmission using laser-scanning microscopy. We report here that syntaxin- 1A is predominantly localized to the plasma membrane, with a small proportion present in an intracellular compartment with a lumenal pH consistent with synaptic vesicles. However, the internal fraction of syntaxin- 1A is excluded from synaptic vesicles that undergo action potential-dependent recycling. These data indicate that the molecular identity of opposing exocytotic membranes is preserved by the sorting of syntaxin- 1A from recycling synaptic vesicles.