SNARE zippering requires activation by SNARE-like peptides in Sec1/Munc18 proteins

Abstract

Soluble -ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze membrane fusion by forming coiled-coil bundles between membrane bilayers. The SNARE bundle zippers progressively toward the membranes, pulling the lipid bilayers into close proximity to fuse. In this work, we found that the +1 and +2 layers in the C-terminal domains (CTDs) of SNAREs are dispensable for reconstituted SNARE-mediated fusion reactions. By contrast, all CTD layers are required for fusion reactions activated by the cognate Sec1/Munc18 (SM) protein or a synthetic Vc peptide derived from the vesicular (v-) SNARE, correlating with strong acceleration of fusion kinetics. These results suggest a similar mechanism underlying the stimulatory functions of SM proteins and Vc peptide in SNARE-dependent membrane fusion. Unexpectedly, we identified a conserved SNARE-like peptide (SLP) in SM proteins that structurally and functionally resembles Vc peptide. Like Vc peptide, SLP binds and activates target (t-) SNAREs, accelerating the fusion reaction. Disruption of the t-SNARE-SLP interaction inhibits exocytosis in vivo. Our findings demonstrated that a t-SNARE-SLP intermediate must form before SNAREs can drive efficient vesicle fusion.

Intracellular Vesicle Fusion Requires a Membrane-Destabilizing Peptide Located at the Juxtamembrane Region of the v-SNARE

“…In contrast, the basal SNAREdriven fusion (without Munc18-1) was not affected by the mutation ( Figures 2C-2E and S2). The selective involvement of the juxtamembrane motif in the SNARE-Munc18-1-mediated fusion reaction is consistent with the discovery that basal SNAREdriven fusion is fundamentally distinct from the SM protein-assisted fusion pathway (Baker et al, 2015;Jiao et al, 2018;Yu et al, 2018). The SNARE-SM-mediated fusion reaction, but not basal SNARE-driven fusion, recapitulates intracellular vesicle fusion Yu et al, 2015Yu et al, , 2018.…”

“…The vesicle-anchored v-SNARE pairs with the target membrane-associated t-SNAREs to form a four-helix trans-SNARE complex, forcing the two membranes into close apposition to fuse (Krä mer and Ungermann, 2011;Schwartz and Merz, 2009;Sö llner et al, 1993). A cognate SM protein activates SNARE zippering and ensures SNARE pairing specificity (Baker et al, 2015;Ma et al, 2013;Shen et al, 2007;Yu et al, 2018).…”

Genetic evidence for an inhibitory role of tomosyn in insulin‐stimulated GLUT4 exocytosis

“…Our previous studies showed that tomosyn's inhibitory activity is dominant over the stimulatory function of Munc18c/Munc18‐3, a key positive regulator of GLUT4 exocytosis 50 . More recently, we discovered that Munc18c uses a SNARE‐like peptide to recognize the C‐terminal domains of t‐SNAREs, 71 which overlap with the tomosyn‐binding site on t‐SNAREs. These observations suggest that tomosyn physically blocks the association of Munc18c with t‐SNAREs.…”

“…The interactions of soluble factors with liposomes were measured using a liposome coflotation assay 60,71,77,80 . Soluble proteins were incubated with liposomes at 4°C with gentle agitation.…”

The N-peptide–binding mode is critical to Munc18-1 function in synaptic exocytosis

“…The biotin-labeled t-SNARE liposomes containing syntaxin-1 and SNAP-25 were anchored to avidin beads and used to pull down rhodamine-labeled v-SNARE liposomes. A VAMP2NTD-TolA chimera was used as the v-SNARE in the docking assay to selectively assess the NTD-mediated docking step (66). The binding reactions were performed at 4°C for 1 h in the absence or presence of 5 M Munc18-1 (WT or clogged).…”