- Email: [email protected]
Membrane Trafficking: Three Steps to Fusion
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responses to other disturbances, and discover the neurophysiological basis for the control. References 1. Daley, M.A., Felix, G., and Biewener, A.A. (2007). Running stability is enhanced by a proximo-distal gradient in joint neuromechanical control. J. Exp. Biol. 210, 383–394. 2. Jayes, A.S., and Alexander, R.McN. (1980). The gaits of chelonians: walking techniques for very low speeds. J. Zool. 191, 353–378. 3. McGeer, T. (1990). Passive dynamic walking. Internat. J. Robotics Res. 9, 62–82.
4. Kubow, T.M., and Full, R.J. (1999). The role of the mechanical system in control: a hypothesis of self-stabilization in hexapedal runners. Phil. Trans. Roy. Soc. B. 354, 849–861. 5. Jindrich, D.L., and Full, R.J. (2002). Dynamic stabilization of rapid hexapedal locomotion. J. Exp. Biol. 205, 2803–2823. 6. Gorassini, M.A., Prochazka, A., Hiebert, G.W., and Gauthier, J.A. (1994). Corrective responses to loss of ground support during walking. I. Intact cats. J. Neurophysiol. 71, 603–610. 7. Daley, M.A., and Biewener, A.A. (2006). Running over rough terrain reveals limb control for intrinsic stability. Proc. Nat. Acad. Sci. USA 103, 15681–15686.
Membrane Trafficking: Three Steps to Fusion Membrane fusion involves the action of members of the SNARE protein family as well as Sec1/Munc18 (SM) proteins, which have been found to interact with SNAREs in three distinct ways. Recent work has established that Munc18-1 directly stimulates fusion and possibly uses all three modes of SNARE interaction. Robert D. Burgoyne1 and Alan Morgan2 Membrane fusion is required during intracellular trafficking to allow vesicles to merge with their target membrane. The accumulation of considerable data over the past decade has firmly established the principle that all intracellular membrane fusion events in the exocytotic and endocytotic pathways use members of the same evolutionarily conserved protein families. One of these groups of proteins is the SNARE family, which comprises vesicular (v-) and target (t-) SNARE isoforms [1]. These proteins have crucial roles in membrane trafficking and it is believed that the assembly of v- and t-SNAREs into a complex is capable of driving membrane fusion [1]. It is clear, however, that additional proteins are required to improve the specificity of SNARE-mediated fusion, to provide regulatory control and, in the case of neurotransmitter release, to dramatically increase the kinetics of the process. Members of the Sec1/Munc18-like (SM) family of proteins function in all SNARE-mediated fusion
events, but their exact roles have not been clear. A full understanding of the action of SM proteins has been complicated by biochemical and structural studies suggesting that different SNARE–SM combinations have different modes of interaction. Also, one of the most well-studied SM proteins is the neuronal exocytotic protein Munc18-1, which, perhaps surprisingly, binds very tightly to a closed conformation of syntaxin that precludes its involvement in fusion [2]: Munc18-1 should, therefore, inhibit fusion, yet it is actually required for neurotransmitter release in vivo [3]. In addition, there has been considerable debate about whether Munc18-1 is involved only in secretory-vesicle docking at the target membrane [4] or additionally in fusion itself [5]. Recent findings now reveal new modes of interaction of Munc18-1 with the assembled SNARE complex [6–8] that are consistent with interactions seen between other SM family members and SNARE proteins and, importantly, these studies have also shown that the Munc18-1–SNARE interaction stimulates the rate of fusion in an
8. Daley, M.A., Usherwood, J.R., Felix, G., and Biewener, A.A. (2006). Running over rough terrain: guinea fowl maintain dynamic stability despite a large unexpected change in substrate height. J. Exp. Biol. 209, 171–187. 9. Seyfarth, A., Geyer, H., and Herr, H. (2003). Swing-leg retraction: a simple control model for stable running. J. Exp. Biol. 206, 2547–2555.
Institute for Integrative and Comparative Biology, University of Leeds, L.C. Miall Building, Leeds LS2 9JT, UK. E-mail: [email protected] DOI: 10.1016/j.cub.2007.02.001
in vitro model system [6]. These new findings go some way towards resolving the apparent lack of conservation in the mode of action of SM proteins and provide new mechanistic insights into a role for Munc18-1 in enhancing the kinetics and specificity of membrane fusion. Genetic studies have established that each step of SNARE-dependent trafficking has an associated SM protein [9]. Our understanding of SM protein function has been hampered, however, by the discovery of three distinct modes of direct interaction with SNARE proteins, two of which have been characterised structurally (Figure 1). The Mode 1 interaction has been observed only with Munc18-1 and involves a tight interaction with monomeric syntaxin1 in its so-called ‘closed’ conformation [2,10], preventing transition of syntaxin1 into its ‘open’ conformation, which is required for binding to other SNAREs. This mode of interaction has been studied using a mutant of syntaxin1 that is constitutively in the open conformation and shows dramatically reduced binding to Munc18-1 in vitro [10]. The Mode 2 interaction has been described for the yeast proteins Sly1p and Vps45p [11,12] and involves binding to the extreme amino terminus of their appropriate syntaxins (Figure 1) but does not prevent assembly of the syntaxins into the SNARE complex. Mutations in Sly1p [13] or Vps45p [14] that prevent this interaction do not abolish Sly1p or Vps45p function in yeast, indicating that Mode 2 interactions may have
Current Biology Vol 17 No 7 R256
A Sly1p–Sed5p
Munc18-1–syntaxin 1a
Syntaxin 1a
Amino terminus of Sed5p Closed syntaxin 1a
Open syntaxin 1a Amino-terminal domain of syntaxin 1a
SNARE core complex
B
Mode 1
Closed syntaxin
SM
Mode 2
Mode 3
SM
Open syntaxin
SM
SNARE complex Current Biology
Figure 1. Structures of SM proteins and syntaxins and modes of SM–syntaxin interactions. (A) Crystal structures are shown for the Munc18-1–syntaxin 1A complex (PDB 1DN1) and a complex between Sly1p and the amino-terminal first 21 residues of Sed5p (PDB 1MQS). Also shown underneath are the structure of the closed syntaxin 1A molecule taken from the Munc18-1–syntaxin 1A complex and a schematic of the open syntaxin 1A molecule based on the structure of the amino-terminal domain of syntaxin 1A (PDB 1BR0) and the core SNARE complex (PDB 1SFC). The structure of the loop connecting the amino- and carboxy-terminal domains of syntaxin 1A is unknown and is shown as a broken line. In all of the figures, the syntaxin is shown in yellow. (B) Schematic representation of the three known modes of binding between SM proteins and syntaxins (see text for details).
a facilitatory but not an essential role. Mode 3 has been described only for yeast Sec1 and involves its binding to assembled complexes of the t-SNAREs and more strongly to the full SNARE complex, but not to its monomeric syntaxin [15,16]. This mode of binding has not been characterised in detail. It would be surprising if the different SM proteins each used distinct modes of interaction and in fact it
has been suggested that Vps45p may use at least two modes of binding [14]. It has not been clear why conserved members of the same protein family would show such distinct protein–protein interactions with a common interacting family. An added complication is that the Mode 1 interaction seen for Munc18-1 would seem to make Munc18-1 an
inhibitor of fusion and yet we know from knock-out studies that Munc18-1 and its orthologues are required for neurotransmitter release. To try to reconcile this apparent contradiction, the Mode 1 interaction has been suggested to allow Munc18-1 to act as a chaperone for syntaxin 1 to promote its transport to the cell surface or prevent its degradation. The interaction with closed syntaxin also cannot wholly account for Munc18-1 function as expression of a constitutively open form of syntaxin (UNC64) in C. elegans supports neurotransmitter release, which requires UNC18 [17]. Syntaxin 1 is present in cells at higher levels than Munc18-1, and it is possible, therefore, that the Mode 1 interaction is a dead-end complex or a mechanism to sequester and inactivate Munc18-1. Some mutations in Munc18-1 that disrupt the Mode 1 binding to syntaxin1 lead to increased neurotransmission [18] and affect late stages in membrane fusion during exocytosis [5]. Significantly, flies expressing these mutations in the Munc18-1 orthologue ROP have impaired viability [18], suggesting that the Mode 1 interaction has a positive role and might perhaps be involved in Munc18-1-mediated vesicle docking. SNARE-mediated fusion has been reconstituted in vitro by mixing proteoliposomes containing either v- or t-SNAREs [1]. In this assay, using the yeast exocytotic SNAREs, it was found that Sec1 stimulated the rate of membrane fusion [16]. This would be consistent with Mode 3 binding by Sec1 and an action on the SNARE complex. Recent work has now examined the effect of Munc18-1 on in vitro fusion mediated by the neuronal exocytotic SNAREs [6]. When added to a mixture containing free v- and t-SNARE liposomes, Munc18-1 had no effect. In contrast, when the liposomes had been pre-incubated at 4 C for 3 hours to allow pre-assembly of the SNARE complex and docking of the liposomes, addition of Munc18-1 then stimulated the rate of fusion up to 20-fold after
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warming to 37 C. Similar observations made for yeast exocytotic proteins [16] point to a similar mode of action in yeast. The stimulation by Munc18-1 was only observed for fusion mediated by the appropriate exocytotic SNARES (syntaxin1, SNAP-25 and VAMP2), revealing mechanistic insights into the overall specificity of membrane fusion. The authors were able to show that this stimulation required pre-assembly of a SNARE complex but did not require Mode 1 binding of Munc18-1 to syntaxin, because Munc18-1 still stimulated fusion in an assay using the open mutant of syntaxin1, which binds poorly to Munc18-1 in vitro. Significantly, the stimulation of fusion by Munc18-1 was abolished when a mutation was introduced into the amino terminus of syntaxin 1 in a conserved residue at position 8, suggesting that the stimulation of fusion required a Mode 2 interaction. Shen et al. [6] were able to demonstrate binding of Munc18-1 to assembled SNARE complexes, and, consistent with the fusion data, this interaction was abolished by the L8A mutation but not the open mutation in syntaxin 1. Other recent work has also identified the existence of Mode 1 and Mode 2 interactions of Munc18-1 with syntaxin 1 and has shown that these interactions can occur both in vitro and also in living cells [7,8]. It is unclear what was different about the binding assay conditions used in the recent papers that allowed the authors to discover this interaction of Munc18-1 with the assembled SNARE complex when so many previous attempts by other labs had failed. Nevertheless, the new results are in agreement with the finding that Munc18-1 can assemble with SNARE complexes on native membranes [19]. Intriguingly, evidence from the minimal fusion assay suggested the existence and functional significance of a third type of interaction [6]. Munc18-1stimulated fusion, but not basal fusion, was reduced by mutations in VAMP2 residues that would be on the surface of the SNARE complex. This suggests that a functionally important interaction
must occur between Munc18-1 and the SNARE complex that is distinct from the Mode 2 interaction and potentially similar to the Mode 3 interaction seen with Sec1. The Mode 3 interaction was independently demonstrated, but the initial attempt to characterise this interaction at a structural level did not allow identification of the specific residues involved [8]. There has been debate in the literature as to whether SM proteins act solely in vesicle docking or additionally in late stages of membrane fusion. Analysis of neuroendocrine cells derived from Munc18-1 knock-out mice has indicated a defect in secretory granule docking [4], although this was not observed for synaptic vesicles in brain synapses [3]. This early defect would, however, mask any additional later roles for Munc18-1 that have been suggested from other studies [5]. Analysis of Munc18-1 mutants had previously suggested that Munc18-1 has both early and late roles in fusion and that these may be independent of the Mode 1 interaction with syntaxin [20]. The new findings support a role for Munc18-1 in late stages accelerating membrane fusion. There are a number of unanswered issues regarding the exact nature of the newly discovered interactions made by Munc18-1. Does the interaction with the amino terminus of syntaxin 1 match the structurally characterised Mode 2 interaction of Sly1p and Vps45p with syntaxins Sed5p and Tlg2p, respectively? What is the structural basis of the functionally important interaction of Munc18-1 with the SNARE complex? Do all of the SM proteins undergo all three interaction modes? Are there any other functionally important modes of interaction? Finally, what is the significance of the distinct interaction modes for the steps of docking and fusion in vivo? Mode 1 binding has only been observed so far for Munc18-1 but, if we assume that SM proteins can indulge in all three modes of SNARE interaction, then we can imagine involvement of SM proteins in three steps leading to membrane fusion: first, vesicle docking at the target
membrane stimulated by the SM protein in its Mode 1 interaction with syntaxin; second, regulated dissociation of the SM protein and formation of the Mode 2 interaction with the amino terminus of syntaxin to target the SM protein to sites of membrane fusion; third, formation of the Mode 3 interaction of the SM protein with the SNARE complex to potentially facilitate SNARE complex assembly and allow acceleration of SNARE-dependent fusion. It is clear that more work needs to be done, particularly in the structural characterisation of Mode 3 binding, but the new findings have provided important clues that will drive the field forward towards a fuller understanding of SM protein function in membrane fusion. References 1. Weber, T., Zemelman, B.V., McNew, J.A., Westermann, B., Gmachl, M., Parlati, F., Sollner, T.H., and Rothman, J.E. (1998). SNAREpins: minimal machinery for membrane fusion. Cell 92, 759–772. 2. Misura, K.M.S., Scheller, R.H., and Weis, W.I. (2000). Three-dimensional structure of the neuronal-Sec1-syntaxin 1a complex. Nature 404, 355–362. 3. Verhage, M., Maia, A.S., Plomp, J.J., Brussaard, A.B., Heeroma, J.H., Vermeer, H., Toonen, R.F., Hammer, R.E., van den Berg, T.K., Missler, M., et al. (2000). Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 287, 864–869. 4. Voets, T., Toonen, R., Brian, E.C., de Wit, H., Moser, T., Rettig, J., Sudhof, T.C., Neher, E., and Verhage, M. (2001). Munc-18 promotes large densecore vesicle docking. Neuron 31, 581–591. 5. Fisher, R.J., Pevsner, J., and Burgoyne, R.D. (2001). Control of fusion pore dynamics during exocytosis by Munc18. Science 291, 875–878. 6. Shen, J., Tareste, D.C., Paumet, F., Rothman, J.E., and Melia, T.J. (2007). Selective activation of cognate SNAREpins by Sec1/Munc18 proteins. Cell 128, 183–195. 7. Rickman, C., Medine, C.N., Bergmann, A., and Duncan, R.R. (2007). Functionally and spatially distinct modes of MUNC18-syntaxin 1 interaction. J. Biol. Chem., epub ahead of print. 8. Dulubova, I., Khvotchev, M., Liu, S., Huryeva, I., Sudhof, T.C., and Rizo, J. (2007). Munc18-1 binds directly to the neuronal SNARE complex. Proc. Natl. Acad. Sci. USA 104, 2697–2702. 9. Jahn, R. (2000). Sec1/Munc18 proteins: mediators of membrane fusion moving to centre stage. Neuron 27, 201–204. 10. Dulubova, I., Sugita, S., Hill, S., Hosaka, M., Fernandez, I., Sudhof, T.C., and Rizo, J. (1999). A conformational switch in syntaxin during exocytosis: role of munc18. EMBO J. 18, 4372–4382. 11. Dulubova, I., Yamaguchi, T., Gao, Y., Min, S.-W., Huryeva, I., Sudhof, T.C., and Rizo, J. (2002). How TIg2p/syntaxin 16 ‘‘snares’’ Vps45. EMBO J. 21, 3620–3631. 12. Bracher, A., and Weissenhorn, W. (2002). Structural basis for the Golgi membrane
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13.
14.
15.
16.
recruitment of Sly1p by Sed5p. EMBO J. 21, 6114–6124. Peng, R., and Gallwitz, D. (2004). Multiple SNARE interactions of an SM protein: Sed5p/Sly1p binding is dispensible for transport. EMBO J. 23, 3939–3949. Carpp, L.N., Ciufo, L.F., Shanks, S.G., Boyd, A., and Bryant, N.J. (2006). The Sec1p/Munc18 protein Vps45p binds its cognate SNARE proteins via two distinct modes. J. Cell Biol. 173, 927–936. Carr, C.M., Grote, E., Munson, M., Hughson, F.M., and Novick, P.J. (1999). Sec1p binds to SNARE complexes and concentrates at sites of secretion. J. Cell Biol. 146, 333–344. Scott, B.L., van Komen, J.S., Irshad, H., Liu, S., Wilson, K.A., and McNew, J.A. (2004). Sec1p directly stimulates
SNARE-mediated membrane fusion in vitro. J. Cell Biol. 167, 75–85. 17. Richmond, J.E., Weimer, R.M., and Jorgensen, E.M. (2001). An open form of syntaxin bypasses the requirement for UNC-13 in vesicle priming. Nature 412, 338–341. 18. Wu, M.N., Littleton, J.T., Bhat, M.A., Prokop, A., and Bellen, H.J. (1998). ROP, the Drosophila sec1 homolog, interacts with syntaxin and regulates neurotransmitter release in a dosage-dependent manner. EMBO J. 17, 127–139. 19. Zilly, F.E., Sorensen, J.B., Jahn, R., and Lang, T. (2006). Munc18-bound syntaxin readily forms SNARE complexes with synaptobrevin in native plasma membranes. PloS. Biol. 4, e330.
20. Ciufo, L.F., Barclay, J.W., Burgoyne, R.D., and Morgan, A. (2005). Munc18-1 regulates early and late stages of exocytosis via syntaxin independent protein interactions. Mol. Biol. Cell 16, 470–482.
The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown Street, P.O. Box 147, Liverpool L69 3BX, UK. E-mail: [email protected], 2 [email protected]
DOI: 10.1016/j.cub.2007.02.006
responses to other disturbances, and discover the neurophysiological basis for the control. References 1. Daley, M.A., Felix, G., and Biewener, A.A. (2007). Running stability is enhanced by a proximo-distal gradient in joint neuromechanical control. J. Exp. Biol. 210, 383–394. 2. Jayes, A.S., and Alexander, R.McN. (1980). The gaits of chelonians: walking techniques for very low speeds. J. Zool. 191, 353–378. 3. McGeer, T. (1990). Passive dynamic walking. Internat. J. Robotics Res. 9, 62–82.
4. Kubow, T.M., and Full, R.J. (1999). The role of the mechanical system in control: a hypothesis of self-stabilization in hexapedal runners. Phil. Trans. Roy. Soc. B. 354, 849–861. 5. Jindrich, D.L., and Full, R.J. (2002). Dynamic stabilization of rapid hexapedal locomotion. J. Exp. Biol. 205, 2803–2823. 6. Gorassini, M.A., Prochazka, A., Hiebert, G.W., and Gauthier, J.A. (1994). Corrective responses to loss of ground support during walking. I. Intact cats. J. Neurophysiol. 71, 603–610. 7. Daley, M.A., and Biewener, A.A. (2006). Running over rough terrain reveals limb control for intrinsic stability. Proc. Nat. Acad. Sci. USA 103, 15681–15686.
Membrane Trafficking: Three Steps to Fusion Membrane fusion involves the action of members of the SNARE protein family as well as Sec1/Munc18 (SM) proteins, which have been found to interact with SNAREs in three distinct ways. Recent work has established that Munc18-1 directly stimulates fusion and possibly uses all three modes of SNARE interaction. Robert D. Burgoyne1 and Alan Morgan2 Membrane fusion is required during intracellular trafficking to allow vesicles to merge with their target membrane. The accumulation of considerable data over the past decade has firmly established the principle that all intracellular membrane fusion events in the exocytotic and endocytotic pathways use members of the same evolutionarily conserved protein families. One of these groups of proteins is the SNARE family, which comprises vesicular (v-) and target (t-) SNARE isoforms [1]. These proteins have crucial roles in membrane trafficking and it is believed that the assembly of v- and t-SNAREs into a complex is capable of driving membrane fusion [1]. It is clear, however, that additional proteins are required to improve the specificity of SNARE-mediated fusion, to provide regulatory control and, in the case of neurotransmitter release, to dramatically increase the kinetics of the process. Members of the Sec1/Munc18-like (SM) family of proteins function in all SNARE-mediated fusion
events, but their exact roles have not been clear. A full understanding of the action of SM proteins has been complicated by biochemical and structural studies suggesting that different SNARE–SM combinations have different modes of interaction. Also, one of the most well-studied SM proteins is the neuronal exocytotic protein Munc18-1, which, perhaps surprisingly, binds very tightly to a closed conformation of syntaxin that precludes its involvement in fusion [2]: Munc18-1 should, therefore, inhibit fusion, yet it is actually required for neurotransmitter release in vivo [3]. In addition, there has been considerable debate about whether Munc18-1 is involved only in secretory-vesicle docking at the target membrane [4] or additionally in fusion itself [5]. Recent findings now reveal new modes of interaction of Munc18-1 with the assembled SNARE complex [6–8] that are consistent with interactions seen between other SM family members and SNARE proteins and, importantly, these studies have also shown that the Munc18-1–SNARE interaction stimulates the rate of fusion in an
8. Daley, M.A., Usherwood, J.R., Felix, G., and Biewener, A.A. (2006). Running over rough terrain: guinea fowl maintain dynamic stability despite a large unexpected change in substrate height. J. Exp. Biol. 209, 171–187. 9. Seyfarth, A., Geyer, H., and Herr, H. (2003). Swing-leg retraction: a simple control model for stable running. J. Exp. Biol. 206, 2547–2555.
Institute for Integrative and Comparative Biology, University of Leeds, L.C. Miall Building, Leeds LS2 9JT, UK. E-mail: [email protected] DOI: 10.1016/j.cub.2007.02.001
in vitro model system [6]. These new findings go some way towards resolving the apparent lack of conservation in the mode of action of SM proteins and provide new mechanistic insights into a role for Munc18-1 in enhancing the kinetics and specificity of membrane fusion. Genetic studies have established that each step of SNARE-dependent trafficking has an associated SM protein [9]. Our understanding of SM protein function has been hampered, however, by the discovery of three distinct modes of direct interaction with SNARE proteins, two of which have been characterised structurally (Figure 1). The Mode 1 interaction has been observed only with Munc18-1 and involves a tight interaction with monomeric syntaxin1 in its so-called ‘closed’ conformation [2,10], preventing transition of syntaxin1 into its ‘open’ conformation, which is required for binding to other SNAREs. This mode of interaction has been studied using a mutant of syntaxin1 that is constitutively in the open conformation and shows dramatically reduced binding to Munc18-1 in vitro [10]. The Mode 2 interaction has been described for the yeast proteins Sly1p and Vps45p [11,12] and involves binding to the extreme amino terminus of their appropriate syntaxins (Figure 1) but does not prevent assembly of the syntaxins into the SNARE complex. Mutations in Sly1p [13] or Vps45p [14] that prevent this interaction do not abolish Sly1p or Vps45p function in yeast, indicating that Mode 2 interactions may have
Current Biology Vol 17 No 7 R256
A Sly1p–Sed5p
Munc18-1–syntaxin 1a
Syntaxin 1a
Amino terminus of Sed5p Closed syntaxin 1a
Open syntaxin 1a Amino-terminal domain of syntaxin 1a
SNARE core complex
B
Mode 1
Closed syntaxin
SM
Mode 2
Mode 3
SM
Open syntaxin
SM
SNARE complex Current Biology
Figure 1. Structures of SM proteins and syntaxins and modes of SM–syntaxin interactions. (A) Crystal structures are shown for the Munc18-1–syntaxin 1A complex (PDB 1DN1) and a complex between Sly1p and the amino-terminal first 21 residues of Sed5p (PDB 1MQS). Also shown underneath are the structure of the closed syntaxin 1A molecule taken from the Munc18-1–syntaxin 1A complex and a schematic of the open syntaxin 1A molecule based on the structure of the amino-terminal domain of syntaxin 1A (PDB 1BR0) and the core SNARE complex (PDB 1SFC). The structure of the loop connecting the amino- and carboxy-terminal domains of syntaxin 1A is unknown and is shown as a broken line. In all of the figures, the syntaxin is shown in yellow. (B) Schematic representation of the three known modes of binding between SM proteins and syntaxins (see text for details).
a facilitatory but not an essential role. Mode 3 has been described only for yeast Sec1 and involves its binding to assembled complexes of the t-SNAREs and more strongly to the full SNARE complex, but not to its monomeric syntaxin [15,16]. This mode of binding has not been characterised in detail. It would be surprising if the different SM proteins each used distinct modes of interaction and in fact it
has been suggested that Vps45p may use at least two modes of binding [14]. It has not been clear why conserved members of the same protein family would show such distinct protein–protein interactions with a common interacting family. An added complication is that the Mode 1 interaction seen for Munc18-1 would seem to make Munc18-1 an
inhibitor of fusion and yet we know from knock-out studies that Munc18-1 and its orthologues are required for neurotransmitter release. To try to reconcile this apparent contradiction, the Mode 1 interaction has been suggested to allow Munc18-1 to act as a chaperone for syntaxin 1 to promote its transport to the cell surface or prevent its degradation. The interaction with closed syntaxin also cannot wholly account for Munc18-1 function as expression of a constitutively open form of syntaxin (UNC64) in C. elegans supports neurotransmitter release, which requires UNC18 [17]. Syntaxin 1 is present in cells at higher levels than Munc18-1, and it is possible, therefore, that the Mode 1 interaction is a dead-end complex or a mechanism to sequester and inactivate Munc18-1. Some mutations in Munc18-1 that disrupt the Mode 1 binding to syntaxin1 lead to increased neurotransmission [18] and affect late stages in membrane fusion during exocytosis [5]. Significantly, flies expressing these mutations in the Munc18-1 orthologue ROP have impaired viability [18], suggesting that the Mode 1 interaction has a positive role and might perhaps be involved in Munc18-1-mediated vesicle docking. SNARE-mediated fusion has been reconstituted in vitro by mixing proteoliposomes containing either v- or t-SNAREs [1]. In this assay, using the yeast exocytotic SNAREs, it was found that Sec1 stimulated the rate of membrane fusion [16]. This would be consistent with Mode 3 binding by Sec1 and an action on the SNARE complex. Recent work has now examined the effect of Munc18-1 on in vitro fusion mediated by the neuronal exocytotic SNAREs [6]. When added to a mixture containing free v- and t-SNARE liposomes, Munc18-1 had no effect. In contrast, when the liposomes had been pre-incubated at 4 C for 3 hours to allow pre-assembly of the SNARE complex and docking of the liposomes, addition of Munc18-1 then stimulated the rate of fusion up to 20-fold after
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warming to 37 C. Similar observations made for yeast exocytotic proteins [16] point to a similar mode of action in yeast. The stimulation by Munc18-1 was only observed for fusion mediated by the appropriate exocytotic SNARES (syntaxin1, SNAP-25 and VAMP2), revealing mechanistic insights into the overall specificity of membrane fusion. The authors were able to show that this stimulation required pre-assembly of a SNARE complex but did not require Mode 1 binding of Munc18-1 to syntaxin, because Munc18-1 still stimulated fusion in an assay using the open mutant of syntaxin1, which binds poorly to Munc18-1 in vitro. Significantly, the stimulation of fusion by Munc18-1 was abolished when a mutation was introduced into the amino terminus of syntaxin 1 in a conserved residue at position 8, suggesting that the stimulation of fusion required a Mode 2 interaction. Shen et al. [6] were able to demonstrate binding of Munc18-1 to assembled SNARE complexes, and, consistent with the fusion data, this interaction was abolished by the L8A mutation but not the open mutation in syntaxin 1. Other recent work has also identified the existence of Mode 1 and Mode 2 interactions of Munc18-1 with syntaxin 1 and has shown that these interactions can occur both in vitro and also in living cells [7,8]. It is unclear what was different about the binding assay conditions used in the recent papers that allowed the authors to discover this interaction of Munc18-1 with the assembled SNARE complex when so many previous attempts by other labs had failed. Nevertheless, the new results are in agreement with the finding that Munc18-1 can assemble with SNARE complexes on native membranes [19]. Intriguingly, evidence from the minimal fusion assay suggested the existence and functional significance of a third type of interaction [6]. Munc18-1stimulated fusion, but not basal fusion, was reduced by mutations in VAMP2 residues that would be on the surface of the SNARE complex. This suggests that a functionally important interaction
must occur between Munc18-1 and the SNARE complex that is distinct from the Mode 2 interaction and potentially similar to the Mode 3 interaction seen with Sec1. The Mode 3 interaction was independently demonstrated, but the initial attempt to characterise this interaction at a structural level did not allow identification of the specific residues involved [8]. There has been debate in the literature as to whether SM proteins act solely in vesicle docking or additionally in late stages of membrane fusion. Analysis of neuroendocrine cells derived from Munc18-1 knock-out mice has indicated a defect in secretory granule docking [4], although this was not observed for synaptic vesicles in brain synapses [3]. This early defect would, however, mask any additional later roles for Munc18-1 that have been suggested from other studies [5]. Analysis of Munc18-1 mutants had previously suggested that Munc18-1 has both early and late roles in fusion and that these may be independent of the Mode 1 interaction with syntaxin [20]. The new findings support a role for Munc18-1 in late stages accelerating membrane fusion. There are a number of unanswered issues regarding the exact nature of the newly discovered interactions made by Munc18-1. Does the interaction with the amino terminus of syntaxin 1 match the structurally characterised Mode 2 interaction of Sly1p and Vps45p with syntaxins Sed5p and Tlg2p, respectively? What is the structural basis of the functionally important interaction of Munc18-1 with the SNARE complex? Do all of the SM proteins undergo all three interaction modes? Are there any other functionally important modes of interaction? Finally, what is the significance of the distinct interaction modes for the steps of docking and fusion in vivo? Mode 1 binding has only been observed so far for Munc18-1 but, if we assume that SM proteins can indulge in all three modes of SNARE interaction, then we can imagine involvement of SM proteins in three steps leading to membrane fusion: first, vesicle docking at the target
membrane stimulated by the SM protein in its Mode 1 interaction with syntaxin; second, regulated dissociation of the SM protein and formation of the Mode 2 interaction with the amino terminus of syntaxin to target the SM protein to sites of membrane fusion; third, formation of the Mode 3 interaction of the SM protein with the SNARE complex to potentially facilitate SNARE complex assembly and allow acceleration of SNARE-dependent fusion. It is clear that more work needs to be done, particularly in the structural characterisation of Mode 3 binding, but the new findings have provided important clues that will drive the field forward towards a fuller understanding of SM protein function in membrane fusion. References 1. Weber, T., Zemelman, B.V., McNew, J.A., Westermann, B., Gmachl, M., Parlati, F., Sollner, T.H., and Rothman, J.E. (1998). SNAREpins: minimal machinery for membrane fusion. Cell 92, 759–772. 2. Misura, K.M.S., Scheller, R.H., and Weis, W.I. (2000). Three-dimensional structure of the neuronal-Sec1-syntaxin 1a complex. Nature 404, 355–362. 3. Verhage, M., Maia, A.S., Plomp, J.J., Brussaard, A.B., Heeroma, J.H., Vermeer, H., Toonen, R.F., Hammer, R.E., van den Berg, T.K., Missler, M., et al. (2000). Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 287, 864–869. 4. Voets, T., Toonen, R., Brian, E.C., de Wit, H., Moser, T., Rettig, J., Sudhof, T.C., Neher, E., and Verhage, M. (2001). Munc-18 promotes large densecore vesicle docking. Neuron 31, 581–591. 5. Fisher, R.J., Pevsner, J., and Burgoyne, R.D. (2001). Control of fusion pore dynamics during exocytosis by Munc18. Science 291, 875–878. 6. Shen, J., Tareste, D.C., Paumet, F., Rothman, J.E., and Melia, T.J. (2007). Selective activation of cognate SNAREpins by Sec1/Munc18 proteins. Cell 128, 183–195. 7. Rickman, C., Medine, C.N., Bergmann, A., and Duncan, R.R. (2007). Functionally and spatially distinct modes of MUNC18-syntaxin 1 interaction. J. Biol. Chem., epub ahead of print. 8. Dulubova, I., Khvotchev, M., Liu, S., Huryeva, I., Sudhof, T.C., and Rizo, J. (2007). Munc18-1 binds directly to the neuronal SNARE complex. Proc. Natl. Acad. Sci. USA 104, 2697–2702. 9. Jahn, R. (2000). Sec1/Munc18 proteins: mediators of membrane fusion moving to centre stage. Neuron 27, 201–204. 10. Dulubova, I., Sugita, S., Hill, S., Hosaka, M., Fernandez, I., Sudhof, T.C., and Rizo, J. (1999). A conformational switch in syntaxin during exocytosis: role of munc18. EMBO J. 18, 4372–4382. 11. Dulubova, I., Yamaguchi, T., Gao, Y., Min, S.-W., Huryeva, I., Sudhof, T.C., and Rizo, J. (2002). How TIg2p/syntaxin 16 ‘‘snares’’ Vps45. EMBO J. 21, 3620–3631. 12. Bracher, A., and Weissenhorn, W. (2002). Structural basis for the Golgi membrane
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The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown Street, P.O. Box 147, Liverpool L69 3BX, UK. E-mail: [email protected], 2 [email protected]
DOI: 10.1016/j.cub.2007.02.006