Fast Vesicle Fusion in Living Cells Requires at Least Three SNARE Complexes

Abstract

Three's the Charm The molecular machinery mediating membrane fusion during secretion from a cell requires a complex of so-called SNARE protein that forms a coiled bundle of four parallel α-helices. Mohrmann et al. (p. 502 , published online 16 September) developed an elegant approach to find out how many SNARE complexes are required to promote secretion of individual secretory vesicles in living chromaffin cells by titrating the ratio of wild-type and mutant SNARE proteins expressed. For fast synchronous release, a minimum of three SNARE complexes per vesicle were required. Fewer SNARE complexes resulted in slower release.

Membrane fusion proteins cooperate to promote rapid secretory vesicle exocytosis from neuroendocrine cells.

Exocytosis requires formation of SNARE [soluble N -ethylmaleimide–sensitive factor attachment protein (SNAP) receptor] complexes between vesicle and target membranes. Recent assessments in reduced model systems have produced divergent estimates of the number of SNARE complexes needed for fusion. Here, we used a titration approach to answer this question in intact, cultured chromaffin cells. Simultaneous expression of wild-type SNAP-25 and a mutant unable to support exocytosis progressively altered fusion kinetics and fusion-pore opening, indicating that both proteins assemble into heteromeric fusion complexes. Expressing different wild-type:mutant ratios revealed a third-power relation for fast (synchronous) fusion and a near-linear relation for overall release. Thus, fast fusion typically observed in synapses and neurosecretory cells requires at least three functional SNARE complexes, whereas slower release might occur with fewer complexes. Heterogeneity in SNARE-complex number may explain heterogeneity in vesicular release probability.

Three's the Charm The molecular machinery mediating membrane fusion during secretion from a cell requires a complex of so-called SNARE protein that forms a coiled bundle of four parallel α-helices. Mohrmann et al. (p. 502 , published online 16 September) developed an elegant approach to find out how many SNARE complexes are required to promote secretion of individual secretory vesicles in living chromaffin cells by titrating the ratio of wild-type and mutant SNARE proteins expressed. For fast synchronous release, a minimum of three SNARE complexes per vesicle were required. Fewer SNARE complexes resulted in slower release.

Membrane fusion proteins cooperate to promote rapid secretory vesicle exocytosis from neuroendocrine cells.

Exocytosis requires formation of SNARE [soluble N -ethylmaleimide–sensitive factor attachment protein (SNAP) receptor] complexes between vesicle and target membranes. Recent assessments in reduced model systems have produced divergent estimates of the number of SNARE complexes needed for fusion. Here, we used a titration approach to answer this question in intact, cultured chromaffin cells. Simultaneous expression of wild-type SNAP-25 and a mutant unable to support exocytosis progressively altered fusion kinetics and fusion-pore opening, indicating that both proteins assemble into heteromeric fusion complexes. Expressing different wild-type:mutant ratios revealed a third-power relation for fast (synchronous) fusion and a near-linear relation for overall release. Thus, fast fusion typically observed in synapses and neurosecretory cells requires at least three functional SNARE complexes, whereas slower release might occur with fewer complexes. Heterogeneity in SNARE-complex number may explain heterogeneity in vesicular release probability.