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Abstract

Fusion of transport vesicles with their target organelles involves specific membrane proteins, SNAREs, which form tight complexes bridging the membranes to be fused. Evidence from yeast and mammals indicates that Sect family proteins act as regulators of membrane fusion by binding to the target membrane SNAREs. In experiments with purified proteins, we now made the observation that the ER to Golgi core SNARE fusion complex could be assembled on syntaxin Sed5p tightly bound to the Sec1- related Sly1p. Sly1p also bound to preassembled SNARE complexes in vitro and was found to be part of a vesicular/target membrane SNARE complex immunoprecipitated from yeast cell lysates. This is in marked contrast to the exocytic SNARE assembly in neuronal cells where high affinity binding of N- Sec1/ Munc-18 to syntaxin 1A precluded core SNARE fusion complex formation. We also found that the kinetics of SNARE complex formation in vitro with either Sly] p-bound or free Sed5p was not significantly different. Importantly, several presumably nonphysiological SNARE complexes easily generated with Sed5p did not form when the syntaxin was first bound to Sly1p. This indicates for the first time that a Sec1 family member contributes to the specificity of SNARE complex assembly.