A Fast Mode of Membrane Fusion Dependent on Tight SNARE Zippering

Abstract

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins have a key role in membrane fusion. It is commonly assumed that pairing of SNARE proteins anchored in opposing membranes overcomes the repulsion energy between membranes, thereby catalyzing fusion. In this study, we have increased the distance between the coiled-coil SNARE motif and the transmembrane domain of the vesicular SNARE synaptobrevin-2 by insertion of a flexible linker to analyze how an increased intermembrane distance affects exocytosis. Synaptobrevin-2 lengthening did not change the frequency of exocytotic events measured at 1 M free calcium but prevented the increase in the secretory activity triggered by higher calcium concentration. Exocytotic events monitored in adrenal chromaffin cells by means of carbon fiber amperometry were classified in two groups according to the rate and extent of fusion pore expansion. Lengthening the juxtamembrane region of synaptobrevin-2 severely reduced the occurrence of rapid single events, leaving slow ones unchanged. It also impaired the increase in the fast-fusion mode that normally follows elevation of intracellular Ca 2ϩ levels. We conclude that mild stimuli trigger slow fusion events that do not rely on a short intermembrane distance. In contrast, a short intermembrane distance mediated by tight zippering of SNAREs is essential to a component of the secretory response elicited by robust stimuli and characterized by rapid dilation of the fusion pore.

Identification of a New Exo-Endocytic Mechanism Triggered by Corticotropin-Releasing Hormone in Mast Cells

“…However, the augmentation of the granular content could be a result of compound fusion (i.e., two or more SG that fuse before exocytosis) (50). Previous analysis of amperometric spikes has indicated that different modes of exocytosis could be reflected in several characteristics, such as the spike half-width or spike rise (39,51,52). Moreover, a previous study suggested that amperometric feet correspond to SG with higher content (35), similar to what we observed in our study (Table II).…”

Distinct Initial SNARE Configurations Underlying the Diversity of Exocytosis

“…Interestingly, the kinetics of exocytosis are markedly altered by the linkers between the SNARE motif and the TMDs of VAMP (66,136,217,316,406) and syntaxin (668), and by the linker between the two SNARE motives in SNAP25 (69,436). Thus the linker regions outside the SNARE motif play an important role in the kinetics of membrane fusion mediated by SNAREs, in line with the idea that SNARE assembly can be induced up to the transmembrane regions (613).…”

“…Exocytosis of LDCVs containing a brain-derived neurotrophic factor (BDNF) is full fusion in dendrites and kiss-and-run fusion in axons (397). SNAREs, and many SNARE-interacting proteins, including Munc18a (173,292), synaptotagmins (579,742), Doc2 (178), SNAP25 (163,436), VAMP2 (66,316), syntaxin (227), SNARE complex (226), and complexin (9,17), affect fusion pore properties (see sect. V).…”

Lipid-Anchored SNAREs Lacking Transmembrane Regions Fully Support Membrane Fusion during Neurotransmitter Release

“…Multiple studies suggest that in addition to the SNARE motifs of synaptobrevin-2, syntaxin-1, and SNAP-25 that mediate SNARE-complex formation, the transmembrane regions (TMRs) of synaptobrevin-2 and syntaxin-1 are essential for membrane fusion and may induce fusion-pore opening (Han et al, 2004; Xu et al, 2005; Deák et al, 2006; Kesavan et al, 2007; Bretou et al, 2008; Lu et al, 2008; Stein et al, 2009; Fdez et al, 2010; Guzman et al, 2010; Ngatchou et al, 2010; Risselada et al, 2011; Shi et al, 2012). In yeast, replacement of the TMR of the synaptobrevin homolog Snc1p with a geranylgeranyl anchor not only blocked membrane fusion during exocytosis, but even transformed Snc1p into an inhibitor of exocytosis (Grote et al, 2000).…”