Munc18-Bound Syntaxin Readily Forms SNARE Complexes with Synaptobrevin in Native Plasma Membranes

Zilly, Felipe E.; Sørensen, Jakob B.; Jahn, Reinhard; Lang, Thorsten

doi:10.1371/journal.pbio.0040330

Cited by 126 publications

(144 citation statements)

References 56 publications

Supporting

Mentioning

126

Contrasting

Unclassified

Order By: Relevance

“…Our direct in vitro binding studies using bacterially expressed GST fusion proteins as well as co-immunoprecipitation results from cell lysates confirmed the in vivo findings. We and others have previously reported that Munc18a also exhibits limited binding to the constitutively open LE mutant of syntaxin 1A in vivo, (15,17), a finding that has been recently extended to include Munc18a interaction with syntaxin 1A in heteromeric Q-SNARE complexes (40) and fully assembled SNARE complexes (24,25). Comparatively, the yeast exocytotic SM protein Sec1p interacts weakly with a closed conformation of Sso1p, favoring binding to Sso1p-Snc2p Q-SNARE complexes (22,23).…”

Section: Discussionmentioning

confidence: 91%

Munc18c Interaction with Syntaxin 4 Monomers and SNARE Complex Intermediates in GLUT4 Vesicle Trafficking

Merrins

Chang

Lam

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

In the process of insulin-stimulated GLUT4 vesicle exocytosis, Munc18c has been proposed to control SNARE complex formation by inactivating syntaxin 4 in a self-associated conformation. Using in vivo fluorescence resonance energy transfer in 3T3L1 adipocytes, co-immunoprecipitation, and in vitro binding assays, we provide data to indicate that Munc18c also associates with nearly equal affinity to a mutant of syntaxin 4 in a constitutively open (unfolded) state (L173A/E174A; LE). To bind to the open conformation of syntaxin 4, we found that Munc18c requires an interaction with the N terminus of syntaxin 4, which resembles Sly1 interaction with the N terminus of ER/Golgi syntaxins. However, both N and C termini of syntaxin 4 are required for Munc18c binding, since a mutation in the syntaxin 4 SNARE domain (I241A) reduces the interaction, irrespective of syntaxin 4 conformation. Using an optical reporter for syntaxin 4-SNARE pairings in vivo, we demonstrate that Munc18c blocks recruitment of SNAP23 to wild type syntaxin 4 yet associates with syntaxin 4 LE -SNAP23 Q-SNARE complexes. Fluorescent imaging of GLUT4 vesicles in 3T3L1 adipocytes revealed that syntaxin 4 LE expressed with Munc18c bypasses the requirement of insulin for GLUT4 vesicle plasma membrane docking. This effect was attenuated by reducing the Munc18c-syntaxin 4 LE interaction with the I241A mutation, indicating that Munc18c facilitates vesicle docking. Therefore, in contradiction to previous models, our data indicates that the conformational "opening" of syntaxin 4 rather than the dissociation of Munc18c is the critical event required for GLUT4 vesicle docking.Soluble N-ethylmaleimide-sensitive factor (NSF) 3 attachment protein receptors (SNAREs) are central to vesicle fusion events in all eukaryotes. SNARE proteins anchored to both transport vesicles and their target membranes overcome the forces necessary for fusion by binding with high affinity in a ternary core complex (1). Although formation of SNARE core complexes is sufficient for membrane fusion (2, 3), essential regulatory proteins of the Sec1/Munc18 (SM) family are believed to control SNARE complex formation. Seven vertebrate SM gene family members (Munc18a/b/c, Sly1, Vps45, and Vps33a/b) have been described, which affect membrane trafficking in distinct subcellular pathways, predominantly through their association with specific syntaxin Q-SNAREs (4).Of the varied SM-syntaxin partners, Munc18c-syntaxin 4 interactions are believed to exert a critically important role in the temporal control of insulin-stimulated GLUT4 storage vesicle exocytosis in muscle and adipose tissue (5-8). The delivery of GLUT4 to the cell surface occurs by distinct signaling processes: 1) GLUT4 vesicle recruitment to the plasma membrane, followed by 2) phosphatidylinositol 3-kinase-dependent vesicle docking and fusion mediated by syntaxin 4-containing SNARE complexes (9, 10). Based on overexpression studies, Munc18c was initially proposed to function as a negative regulator of GLUT4 vesicle translocation in 3T3L1 ad...

show abstract

Section: Discussionmentioning

confidence: 91%

Munc18c Interaction with Syntaxin 4 Monomers and SNARE Complex Intermediates in GLUT4 Vesicle Trafficking

Merrins

Chang

Lam

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…1). Furthermore, evidence is emerging that Munc18-1 can interact with SNARE complexes (21,22,26,27) and that the Stx1a N terminus is important for this interaction (21,22). We surmised that an N-peptide interaction can occur in Munc18-1 and that this would explain the requirement for the N-peptide in SNARE complex interactions with Munc18-1.…”

Section: Munc18-1 Has An N-peptide Binding Sitementioning

confidence: 89%

“…Sly1p (14,20,29), Vps45p (24,30), and Munc18c (19) bind to an N-peptide on their cognate Stxs, and this interaction can accommodate binding to monomeric Stxs as well as SNARE complexes. Evidence is emerging to show that Munc18-1 also binds SNARE complexes (21,22,26,27), as well as monomeric Stx1a. The interaction between Munc18-1 and the SNARE complex relies on the Stx1a N-peptide (21,22).…”

Section: Discussionmentioning

confidence: 99%

Structure of the Munc18c/Syntaxin4 N-peptide complex defines universal features of the N-peptide binding mode of Sec1/Munc18 proteins

Latham

Gee

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

130

148

View full text Add to dashboard Cite

Sec1/Munc18 proteins (SM proteins) bind to soluble NSF attachment protein receptors (SNAREs) and play an essential role in membrane fusion. Divergent modes of regulation have been proposed for different SM proteins indicating that they can either promote or inhibit SNARE assembly. This is in part because of discrete modes of binding that have been described for various SM/SNARE complexes. One mode suggests that SM proteins bind only to Syntaxins (Stx) preventing SNARE assembly, whereas in another they facilitate SNARE assembly and bind to SNARE complexes. The mammalian cell surface SM protein Munc18c binds to an N-peptide in Stx4, and this is compatible with its interaction with SNARE complexes. Here we describe the crystal structure of Munc18c in complex with the Stx4 N-peptide. This structure shows remarkable similarity with a yeast complex indicating that the mode of binding, which can accommodate SNARE complexes, is highly conserved throughout evolution. Modeling reveals the presence of the N-peptide binding mode in most but not all yeast and mammalian SM/Stx pairs, suggesting that it has coevolved to fulfill a specific regulatory function. It is unlikely that the N-peptide interaction alone accounts for the specificity in SM/SNARE binding, implicating other contact surfaces in this function. Together with other data, our results support a sequential two-state model for SM/SNARE binding involving an initial interaction via the Stx N-peptide, which somehow facilitates a second, more comprehensive interaction comprising other contact surfaces in both proteins. crystallography ͉ protein:protein interactions ͉ vesicle trafficking I ntracellular trafficking relies on membrane fusion, a tightly regulated process that requires the formation of a coiled coil complex between helical motifs originating from soluble NSF attachment protein receptor (SNARE) proteins on vesicle and target membranes (1-3). Sec1/Munc18 proteins (SM proteins) play a fundamental role in this process by interacting with SNARE protein(s) and regulating the formation of SNARE complexes (4, 5). SM proteins have been identified in organisms from yeast to humans, and loss-of-function mutants lead to severe impairment in vesicle fusion (6).Despite their obvious importance, the nature of SNARE regulation by SM proteins remains controversial, in that both positive and negative regulatory roles have been reported (6, 7). Possibly the most compelling evidence in support of negative regulation is the observation that the mammalian Syntaxin1a (Stx1a) SNARE protein involved in synaptic neurotransmission exists in both an ''open'' (SNARE complex-compatible) and a ''closed'' (SNARE complexincompatible) conformation (8, 9). The crystal structure of Stx1a in complex with the SM protein Munc18-1 (Munc18a, nSec1) reveals that Munc18-1 embraces a closed conformation preventing Stx1a from forming the SNARE complex required for vesicle fusion (10). On the other hand, deletion of Munc18-1 results in impaired exocytosis in mouse neurons and chromaffin cells (11, 12),...

show abstract

“…Indeed, recent experiments showed that synaptobrevin can displace Munc18-1 from membrane sheets by binding to the other SNAREs (Zilly et al, 2006), and Munc18-1 was found to accelerate vesicle fusion in vitro (Shen et al, 2007). Thus, Munc18-1 might interact with SNAREs through two separate mechanisms, one of which does not prevent, but might actually promote, SNARE complex formation (Rickman et al, 2007;Shen et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Munc18-1: Sequential Interactions with the Fusion Machinery Stimulate Vesicle Docking and Priming

et al. 2007

View full text Add to dashboard Cite

Exocytosis of secretory or synaptic vesicles is executed by a mechanism including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. Munc18-1 is a part of this fusion machinery, but its role is controversial because it is indispensable for fusion but also inhibits the assembly of purified SNAREs in vitro. This inhibition reflects the binding of Munc18-1 to a closed conformation of the target-SNARE syntaxin1. The controversy would be solved if binding to closed syntaxin1 were shown to be stimulatory for vesicle fusion and/or additional essential interactions were identified between Munc18-1 and the fusion machinery. Here, we provide evidence for both notions by dissecting sequential steps of the exocytotic cascade while expressing Munc18 variants in the Munc18-1 null background. In Munc18-1 null chromaffin cells, vesicle docking is abolished and syntaxin levels are reduced. A mutation that diminished Munc18 binding to syntaxin1 in vitro attenuated the vesicle-docking step but rescued vesicle priming in excess of docking. Conversely, expressing the Munc18-2 isoform, which also displays binding to closed syntaxin1, rescued vesicle docking identical with Munc18-1 but impaired more downstream vesicle priming steps. All Munc18 variants restored syntaxin1 levels at least to wild-type levels, showing that the docking phenotype is not caused by syntaxin1 reduction. None of the Munc18 variants affected vesicle fusion kinetics or fusion pore duration. In conclusion, binding of Munc18-1 to closed syntaxin1 stimulates vesicle docking and a distinct interaction mode regulates the consecutive priming step.

show abstract

Munc18-Bound Syntaxin Readily Forms SNARE Complexes with Synaptobrevin in Native Plasma Membranes

Cited by 126 publications

References 56 publications

Munc18c Interaction with Syntaxin 4 Monomers and SNARE Complex Intermediates in GLUT4 Vesicle Trafficking

Munc18c Interaction with Syntaxin 4 Monomers and SNARE Complex Intermediates in GLUT4 Vesicle Trafficking

Structure of the Munc18c/Syntaxin4 N-peptide complex defines universal features of the N-peptide binding mode of Sec1/Munc18 proteins

Munc18-1: Sequential Interactions with the Fusion Machinery Stimulate Vesicle Docking and Priming

Contact Info

Product

Resources

About