COMMENTARY Testing the SNARE/SM protein model of membrane fusion Taulant Bacaja, Zhiping P. Panga, and Thomas C. Südhofa,b,1 aDepartment of Molecular and Cellular Physiology and bHoward Hughes Medical Institute, Stanford University, Stanford, CA 94305-5453 n eukaryotic cells, the budding and Syntaxin domain structure A N-terminal fusion of membranes mediates di- peptide Habc-domain SNARE motif I verse but essential processes, ranging Ha Hb Hc TMR from cell division to organelle bio- genesis to neurotransmitter secretion. All intracellular membrane fusion except for B SNARE/SM protein fusion machinery mitochondrial fusion is driven by SNARE 1 Synapto- 2 and SM (Sec1/Munc18-like) proteins brevin/ (1–3). Membrane fusion has been particu- VAMP larly intensely studied for neurotransmitter Habc- SNAP-25 Syntaxin secretion. In neurotransmitter secretion, domain ? fusion is mediated by the plasma mem- brane SNARE proteins syntaxin and syn- N-peptide Munc18 aptosomal-associated protein 25 (SNAP- 25), the vesicle SNARE protein synapto- ? brevin/vesicle-associated membrane 4 3 protein (VAMP), and the SM protein Munc18-1 (Fig. 1 A and B). Mechanisti- Ca2+ cally, SNARE proteins are thought to fuel fusion by forming a transcomplex between the vesicle and target membranes; in this complex, progressive zippering of a four- Fusion-pore opening Fusion competent helical bundle formed by the SNARE motifs of SNARE proteins forces the fus- ing phospholipid membranes into close C Mechanistic test of N-peptide function proximity, thereby destabilizing their sur- 1 Synapto- 3 faces (1–3). SM proteins are essential brevin/ VAMP Syntaxin coagonists of SNARE proteins in fusion in N-peptide + that all intracellular SNARE-dependent Munc18 Habc-domain fusion reactions require an SM protein. SNAP-25 At least for fusion during neurotrans- mitter secretion, syntaxin constitutes the TolA central organizer that is composed of multiple domains: a conserved N-terminal Syntaxin Fusion competent unstructured peptide, an N-terminal Habc SNARE-motif domain, a SNARE motif, and a C-terminal + TMR transmembrane region (Fig. 1A). Syntaxin Fig. 1. SNARE/SM protein function in vesicle fusion. (A) Domain structure of syntaxins, which are assumes two conformations: a closed con- composed of a conserved ≈20-residue N-terminal sequence, an Habc domain containing three α-helices, formation outside of the SNARE complex a ≈60-residue SNARE motif, and a transmembrane region (TMR). (B) Working model of membrane fusion fi in which the Habc domain folds back onto mechanism of SNARE/SM proteins, exempli ed by synaptic SNARE and SM proteins: (1) synaptic SNAREs the SNARE motif, and an open confor- [synaptobrevin-2 (Syb2/VAMP), syntaxin, and SNAP-25] before SNARE complex assembly, with the SM mation in the SNARE complex with a protein Munc18 bound to the closed conformation of syntaxin; (2) initiation of SNARE complex with opening of syntaxin and continued binding of Munc18 to the syntaxin N-peptide; (3) partial zippering mobile Habc domain (4, 5) (Fig. 1B). Both conformations bind to Munc18, but only of SNARE complexes; (4) completion of the SNARE complex/Munc18 assembly opens the fusion pore. (C) Diagram of the key experiment performed by Rathore et al. (13): an in vitro and in vivo assay in which Munc18 binding to open syntaxin requires – the functionality of two complementary syntaxin mutants was examined when present alone or ex- the N-terminal syntaxin peptide (6 8). pressed simultaneously: in the first syntaxin mutant the SNARE motif was replaced by an unrelated Although both Munc18/syntaxin binding α-helix from the bacterial protein TolA, whereas in the second syntaxin mutant the N-peptide and Habc modes are known to be essential for fusion domain were deleted. The two syntaxin mutants are individually unable to support fusion but when in vivo (9–12), how syntaxin orchestrates both mutant proteins are present simultaneously, liposome fusion is restored. fusion remains unclear. Using elegant in vitro liposome fusion and in vivo Caeno- rhabditis elegans experiments, Rathore In a first set of experiments, Rathore et al. (13) now address the critical question et al. (13) use an in vitro liposome fusion Author contributions: T.B., Z.P.P., and T.C.S. wrote the of whether the N-terminal peptide of assay that monitors lipid mixing to confirm paper. syntaxins acts autonomously in fusion, previous data (14) showing that Munc18 The authors declare no conflict of interest. independent of its anchorage to syntaxin, is essential for efficient liposome fusion See companion article on page 22399. or whether it is required to be coupled and that the syntaxin N-peptide is required, 1To whom correspondence should be addressed. E-mail: to syntaxin. whereas the Habc domain is dispensable. [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1017268108 PNAS | December 28, 2010 | vol. 107 | no. 52 | 22365–22366 Downloaded by guest on September 28, 2021 Then, to test whether the N-peptide acts as (13), as elegant as they are, is that it re- syntaxin N-peptide functions to recruit SM a modular sequence in fusion, Rathore mains unclear whether the results are proteins to the vicinity of assembling et al. translocate the N-peptide from syn- transferable to physiological membrane SNARE complexes (1–3). The importance taxin to the t-SNARE SNAP-25 or to fusion. Even if content-mixing assays of of the article lies not only in the persua- Munc18, or use a soluble version of the fusion had been included, liposome fusion siveness of the evidence but also in the N-peptide. They find that only the hybrid assays still do not completely test the po- approach, which consists of a combination protein composed of the syntaxin N- tential role of the Habc domain in orga- of in vitro liposome fusion with in vivo peptide and SNAP-25 supports liposome nizing the sites of fusion, let alone analyze tests of the resulting conclusions. Like any fusion in the presence of a syntaxin lacking interesting study, the results of Rathore the N-peptide. Furthermore, Rathore et al. (13) also raise further questions. et al. (13) create a syntaxin mutant in which The syntaxin N-peptide is Among many fascinating issues, four stand the SNARE motif is replaced by an α-helix out. First, why do fusion reactions univer- from the bacterial protein TolA. This functionally autonomous sally require SM proteins; and what is their mutant or a syntaxin mutant lacking the in vivo, as long as it is function (1)? Second, is the SM protein N-peptide and Habc domain separately recruitment mechanism showcased here are unable to support liposome fusion, anchored in the target universally applicable to all SNARE- but when both mutants are present simul- mediated fusion events, as suggested by taneously, liposome fusion is restored membrane. the presence of a similar SM protein (Fig. 1C). Finally, Rathore et al. (13) en- binding mode in endoplasmic reticulum gineer a syntaxin with a cleavable N ter- and endosomal syntaxins in which it was minus and incubate this with Munc18 and the function of syntaxin in orchestrating first discovered (15, 16), or do some fusion SNAREs at 4 °C, followed by cleavage of the speed and topology of fusion. Re- reactions, such as those involving the the N-peptide. When incubated at 37 °C, alizing this limitation, Rathore et al. (13) HOPS complex (17), use a different this preparation undergoes fusion, sug- perform a second set of experiments, as- mechanism of SM protein recruitment? gesting that the N-peptide is not neces- saying rescue of neurotransmitter secre- Third, the Habc domain is highly conserved sary when the fusion reaction is activated at tion in a syntaxin mutant of C. elegans.In in syntaxins but dispensable for liposome 37 °C. Together, these experiments show a beautiful demonstration of the power of fusion (13, 14)—does this mean that the that the syntaxin N-peptide does not need genetics, they demonstrate that the com- Habc domain is not required for fusion in to be on syntaxin or a SNARE protein bination of the two complementary syn- vivo to perform a “mere” regulatory role for liposome fusion but has to be on the taxin mutants (the mutant in which the without participation in the fusion mech- target membrane close to the t-SNARE SNARE motif is replaced by an unrelated anism, or has no function at all? Fourth, at proteins, suggesting that the N-peptide α-helix, and the mutant in which the least during neurotransmitter secretion acts independently and autonomously but N-peptide and Habc domain are deleted) syntaxin does more than fusion—it shapes in conjunction with t-SNARE proteins. can partially rescue neurotransmission in the kinetics of secretion and interacts with Moreover, the N-peptide cleavage experi- the neuromuscular junctions of syntaxin- Ca2+ channels (18–20). How do the syn- ments suggest that the N-peptide acts deficient C. elegans (Fig. 1C); expression taxin domains studied here relate to these before full SNARE complex assembly, of each syntaxin mutant separately fails to significant syntaxin functions? Addressing although the experiment does not reveal rescue. This important experiment con- these questions will require multidisci- whether the same is true for the SM pro- clusively shows that the syntaxin N-peptide plinary approaches similar to those re- tein Munc18, whose precise mode of action is functionally autonomous in vivo, as long ported by Rathore et al. (13) and will be remains a mystery. as it is anchored in the target membrane. essential for further progress in un- A potential problem with the liposome The findings of Rathore et al. (13) derstanding membrane fusion beyond the experiments described by Rathore et al. provide the best evidence to date that the identification of its essential components.