Syntaxin/Munc18 Interactions in the Late Events during Vesicle Fusion and Release in Exocytosis

Graham, Margaret E.; Barclay, Jeff W.; Burgoyne, Robert D.

doi:10.1074/jbc.m400827200

Cited by 62 publications

(63 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…4A indicates that the apparent efficiency (E A ) of cYFP-Munc18c in complex with CFP-syntaxin 4 LE was mildly reduced from that measured with CFP-syntaxin 4. By contrast, the apparent efficiency of energy transfer between Munc18a and syntaxin 1A LE was ϳ50% lower than that measured for syntaxin 1A, a result consistent with the strong reduction in affinity conferred by the syntaxin 1A LE mutation reported in other studies (17,29). To further (Fig.…”

Section: Modeling Of Syntaxin 4 Structure and Generation Of A Constitsupporting

confidence: 87%

“…These results establish that FRET between cYFP-Munc18c and CFP-syntaxin 4 is specific to the bimolecular interaction in 3T3L1 adipocytes and sensitive to changes in Munc18c-syntaxin 4 binding affinity. Notably, CFP-syntaxin 4 localized to the plasma membrane when coexpressed with cYFPMunc18c R240L , suggesting that syntaxin 4 does not require a direct interaction with Munc18c for plasma membrane target- Our FRET experiments in adipocytes described above demonstrated a significant interaction of Munc18c with syntaxin 4 LE , which contrasts with the strongly reduced interaction reported for the LE mutant of syntaxin 1A with Munc18a in vitro and in vivo (17,29). This indicates that divergence occurs among vertebrate SM-syntaxin protein interactions.…”

Section: Modeling Of Syntaxin 4 Structure and Generation Of A Constitmentioning

confidence: 66%

“…A mutation in the syntaxin 1A linker helix (L165A/E166A; LE) has been reported to destabilize the N-terminal helices from interaction with the SNARE motif and result in a constitutively open syntaxin 1A conformation (15). This open conformation impairs Munc18a binding to syntaxin 1A, strongly facilitating its interaction with other SNAREs (3,17,29,34). From the structural prediction (Fig.…”

Section: Modeling Of Syntaxin 4 Structure and Generation Of A Constitmentioning

confidence: 99%

“…FRET between Munc18c and syntaxin 41 was not different from wild type syntaxin 4, indicating that Munc18c probably utilizes residues conserved between the two syntaxins to bind the SNARE domain. Mutation of syntaxin 1A at two conserved residues, Ile 209 and Ile 233 , has been reported to reduce binding with SNAP25 and Munc18a, respectively (17,29,37,38). These two amino acids lie within the group of hydrophobic residues that form the interior of the fully assembled SNARE complex.…”

Section: Modeling Of Syntaxin 4 Structure and Generation Of A Constitmentioning

confidence: 99%

See 3 more Smart Citations

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: Modeling Of Syntaxin 4 Structure and Generation Of A Constitsupporting

confidence: 87%

Section: Modeling Of Syntaxin 4 Structure and Generation Of A Constitmentioning

confidence: 66%

Section: Modeling Of Syntaxin 4 Structure and Generation Of A Constitmentioning

confidence: 99%

Section: Modeling Of Syntaxin 4 Structure and Generation Of A Constitmentioning

confidence: 99%

See 2 more Smart Citations

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

“…All electron density maps were generated with a 3.2-Å resolution cutoff. another study showed that Syx1a LE is more susceptible to proteolysis (12). Direct structural data for the Munc18a-Syx1a LE complex have been lacking, however, and our data show that the LE mutant can adopt a closed conformation that tightly binds Munc18a.…”

Section: Resultsmentioning

confidence: 48%

Syntaxin1a variants lacking an N-peptide or bearing the LE mutation bind to Munc18a in a closed conformation

Colbert

Hattendorf

Weiß

et al. 2013

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

View full text Add to dashboard Cite

In neurons, soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins drive the fusion of synaptic vesicles to the plasma membrane through the formation of a four-helix SNARE complex. Members of the Sec1/Munc18 protein family regulate membrane fusion through interactions with the syntaxin family of SNARE proteins. The neuronal protein Munc18a interacts with a closed conformation of the SNARE protein syntaxin1a (Syx1a) and with an assembled SNARE complex containing Syx1a in an open conformation. The N-peptide of Syx1a (amino acids 1-24) has been implicated in the transition of Munc18a-bound Syx1a to Munc18a-bound SNARE complex, but the underlying mechanism is not understood. Here we report the X-ray crystal structures of Munc18a bound to Syx1a with and without its native N-peptide (Syx1aΔN), along with small-angle X-ray scattering (SAXS) data for Munc18a bound to Syx1a, Syx1aΔN, and Syx1a L165A/E166A (LE), a mutation thought to render Syx1a in a constitutively open conformation. We show that all three complexes adopt the same global structure, in which Munc18a binds a closed conformation of Syx1a. We also identify a possible structural connection between the Syx1a N-peptide and SNARE domain that might be important for the transition of closed-to-open Syx1a in SNARE complex assembly. Although the role of the N-peptide in Munc18a-mediated SNARE complex assembly remains unclear, our results demonstrate that the N-peptide and LE mutation have no effect on the global conformation of the Munc18a-Syx1a complex. membrane trafficking | SM proteins | protein crystallography N eurons communicate across specialized intercellular junctions called synapses. Arrival of an action potential in a presynaptic neuron triggers a calcium (Ca 2+ ) influx, which leads to the exocytosis of presynaptic vesicles and release of neurotransmitters for uptake via receptors in the postsynaptic neuron. Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins drive the fusion of neurotransmitter-containing vesicles to the presynaptic plasma membrane, a critical step in Ca 2+ -triggered exocytosis. Three neuronal SNARE proteinssyntaxin 1a (Syx1a) and synaptosomal-associated protein of 25 kDa (SNAP25), located on the synaptic membrane, and vesicleassociated membrane protein 2 (VAMP2, also called synaptobrevin) combine to form a four-helix bundle, a process that drives membrane fusion. Syx1a contains an N-terminal regulatory region in addition to its C-terminal SNARE domain and adopts two conformations: a closed conformation, in which a three-helix bundle (designated Habc) in the N-terminal region binds intramolecularly to the SNARE domain, and an open conformation in which the SNARE domain binds SNAP25 and VAMP2 to form the ternary SNARE complex.The neuronal Sec1/Munc18a (SM)-like protein Munc18a is essential for synaptic vesicle fusion and associates with the neuronal SNARE proteins in at least two distinct modes. Munc18a

show abstract

An organelle proteomic method to study neurotransmission‐related proteins, applied to a neurodevelopmental model of schizophrenia

Vercauteren

Flores

et al. 2007

Proteomics

View full text Add to dashboard Cite

Limited information is currently available on molecular events that underlie schizophrenia-like behaviors in animal models. Accordingly, we developed an organelle proteomic approach enabling the study of neurotransmission-related proteins in the prefrontal cortex (PFC) of postpubertal (postnatal day 60 (PD60)) neonatally ventral hippocampal (nVH) lesioned rats, an extensively used neurodevelopmental model of schizophrenia-like behaviors. The PFC was chosen because of its purported role in the etiology of the disease. Statistical analysis of 392 reproducible spots on 2-D organelle proteomic patterns revealed significant changes in intensity of 18 proteinous spots in plasma membrane-enriched fractions obtained from postpubertal nVH lesioned rats compared to controls. Mass spectrometric analysis and database searching allowed the identification of a single protein in each of the nine differential spots, including proteins of low abundance, such as neurocalcin delta. Most of the identified dysregulated proteins, including clathrin light chain B, syntaxin binding protein 1b and visinin-like protein 1 are known to be linked to various neurotransmitter systems and to play key roles in plasma membrane receptor expression and recycling as well as synaptic vesicle exocytosis/recycling. Organelle proteomic approaches have hence proved to be most useful to identify key proteins linked to a given behavior in animal models of brain diseases.

show abstract

Syntaxin/Munc18 Interactions in the Late Events during Vesicle Fusion and Release in Exocytosis

Cited by 62 publications

References 55 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

Syntaxin1a variants lacking an N-peptide or bearing the LE mutation bind to Munc18a in a closed conformation

An organelle proteomic method to study neurotransmission‐related proteins, applied to a neurodevelopmental model of schizophrenia

Contact Info

Product

Resources

About