Assembly of SNARE proteins between opposing membranes mediates fusion of synthetic lipo-somes, but it is unknown whether SNAREs act during exocytosis at the moment of Ca²⁺ increase, providing the molecular force for fusion of secretory vesicles. Here, weshowthatexecution of pre- and postfusional Steps during chromaffin granule exocytosis depends crucially on a short molecular distance between the complex-forming SNARE motif and the transmembrane anchor of the vesicular SNARE protein synaptobrevin II. Extending the juxtamembrane region of synaptobrevin by insertion of flexible "linkers" reduces priming of granules, delays initiation of exocytosis upon stepwise elevation of intracellular calcium, attenuates fluctuations of early fusion pores, and slows rapid expansion of the pore in a linker-length dependent fashion. These observations provide evidence that v-SNARE proteins drive Ca²⁺-triggered membrane fusion at millisecond time scale and support a model wherein continuous molecular pulling by SNAREs guides the vesicle throughout the consecutive stages of exocytosis
