Rapid and efficient fusion of phospholipid vesicles by the α-helical core of a SNARE complex in the absence of an N-terminal regulatory domain

Abstract

. We find that the isolated core of a SNARE complex efficiently fuses artificial bilayers and does so faster than full length SNAREs. Unexpectedly, a dramatic increase in speed results from removal of the N-terminal domain of the t-SNARE syntaxin, which does not affect the rate of assembly of v-t SNARES. In the absence of this negative regulatory domain, the half-time for fusion of an entire population of lipid vesicles by isolated SNARE cores (Ϸ10 min) is compatible with the kinetics of fusion in many cell types. Many genetic and biochemical experiments have implicated SNAREs in the overall process of membrane fusion (1-6). The finding that isolated SNARE proteins can efficiently fuse lipid bilayers directly establishes that they are the basic machinery that merges membranes (7), a conclusion recently confirmed in an elegant study using permeabilized cells (8) and by the demonstration of contents mixing during SNARE-dependent fusion [see the accompanying paper by Nickel et al. (9)]. This principle is underscored by the internal architecture of the SNARE complex, whose ''core'' consists of four parallel ␣-helices packed into a single bundle with their membrane anchors emerging together from one end of the assembled SNARE complex (10-14).The structure of the cytoplasmic core domain of a SNARE complex is reminiscent of the structure of the extracellularly localized core region of many viral fusion proteins in what is thought to correspond to a postfusogenic conformation (15,16). This suggests a general principle for membrane fusion, in which pin-like structures bridging two membranes promote their fusion. However, no core complex, whether cellular or viral, has actually been shown to be fusogenic. Here, we report such evidence with a core domain of SNARE proteins. Materials and MethodsPlasmid Construction, Protein Expression, and Purification. The following v-SNARE and t-SNARE complexes were bacterially expressed and purified by nickel affinity chromatography: VA MP2His 6 ; syntaxin1͞His 6 SNA P-25 (SYN͞SNA P-25); thrombin-cleavable syntaxin1͞His 6 SNAP-25 (tcSYN͞SNAP-25); syntax in1͞thrombin-cleavableHis 6 SNA P-25 (SYN͞ tcSNAP-25); and thrombin-cleavable syntaxin1͞thrombin-cleavableHis 6 SNAP-25 (tcSYN͞tcSNAP-25). For detailed information, see the supplemental material on the PNAS web site, www.pnas.org.Protein Reconstitution into Liposomes and Thrombin Cleavage of t-SNARE Liposomes. Both VAMP2 and t-SNARE complexes were reconstituted into liposomes as described (7). Once proteoliposomes were harvested, tcSYN͞SNAP-25 liposomes were first treated with human thrombin (Sigma, catalogue number T-1063) at 0.02 units͞l for 2 hours at room temperature and subsequently were inhibited with 2 mM 4-(2-aminoethyl)benzenesulfonyl f luoride (Calbiochem). SYN͞tcSNA P-25, tcSYN͞ tcSNAP-25, or SYN͞SNAP-25 containing proteoliposomes were treated with 0.04 units͞l thrombin (Sigma) for 4 hours at 37°C and subsequently were inhibited with 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride. As a control, thrombin was preinactivated by fir...

Asymmetric phospholipid distribution drives in vitro reconstituted SNARE-dependent membrane fusion

“…1 A, open circles). As previously documented for other v-and t-SNARE pairs, this time frame and extent of membrane increase in fluorescence is characteristic of membrane fusion (19)(20)(21)(22).…”

“…In vitro fusion reactions using reconstituted neuronal SNARE proteins are typically performed by using liposomes composed of PC plus an additional 15-25% PS (19,20,22,23). The addition of PA resulted in a concentrationdependent increase in VAMP2 incorporation, whereas PA had no significant effect on the efficiency of Stx4 incorporation ( Fig.…”

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

“…Munc18a/nSec1, a cytosolic protein, binds to the closed form of syntaxin1 and prevents assembly of the ternary SNARE complex [50,51]. Membrane fusion is promoted when the N-terminal domain of syntaxin1 is removed [39]. To determine the function of the Nterminal domain of syntaxin4, in the current study we developed two truncation mutants of syntaxin4 in which half or the entire N-terminal domain was removed.…”

“…The N-terminal domain of syntaxin1 was shown to exhibit an inhibitory role in membrane fusion [39]. When co-expressed with SNAP-25, syntaxin4 was less fusogenic than syntaxin1 H3 that does not contain its N-terminal domain (compare first column and third column in Fig.…”

VAMP subfamilies identified by specific R-SNARE motifs

“…N-terminus extensions, modulating the formation and/or disassembly of the fusion complex, are quite common among SNAREs (Dietrich et al, 2003). In fact, the t-SNAREs syntaxins share a N-terminal H A H B H C domain necessary for viability and capable of intermolecular inhibition by binding to the Q-SNARE CCD (Nicholson et al, 1998;Parlati et al, 1999;Munson et al, 2000). Moreover, the v-SNAREs longins are characterized by the LD with profilin-like structure (Gonzalez et al, 2001;Tochio et al, 2001); this domain is capable to regulate membrane fusion (Martinez-Arca et al, 2000 and2001), as subcellular targeting (Hasegawa et al, 2003) by interacting with the clathrin adaptor AP-3 in the case of TI-VAMP/VAMP7 (Martinez-Arca et al, 2003), and protein palmitoylation in the case of Ykt6p (Dietrich et al, 2004).…”