scholarly journals Autoinhibition of SNARE complex assembly by a conformational switch represents a conserved feature of syntaxins

2010 ◽  
Vol 38 (1) ◽  
pp. 209-212 ◽  
Author(s):  
Chris MacDonald ◽  
Mary Munson ◽  
Nia J. Bryant
Keyword(s):  
Membrane Fusion ◽  
Membrane Trafficking ◽  
Snare Complex ◽  
Cellular Transport ◽  
Protein Families ◽  
Sm Proteins ◽  
Complex Assembly ◽  
Receptor Complexes ◽  
Snare Motif ◽  
Sm Protein

Regulation and specificity of membrane trafficking are required to maintain organelle integrity while performing essential cellular transport. Membrane fusion events in all eukaryotic cells are facilitated by the formation of specific SNARE (soluble N-ethylmaleimide-sensitive fusion proteinattachment protein receptor) complexes between proteins on opposing lipid bilayers. Although regulation of SNARE complex assembly is not well understood, it is clear that two conserved protein families, the Sx (syntaxin) and the SM (Sec1p/Munc18) proteins, are central to this process. Sxs are a subfamily of SNARE proteins; in addition to the coiled-coil SNARE motif, Sxs possess an N-terminal, autonomously folded, triple-helical (Habc) domain. For some Sxs, it has been demonstrated that this Habc domain exerts an autoinhibitory effect on SNARE complex assembly by making intramolecular contacts with the SNARE motif. SM proteins regulate membrane fusion through interactions with their cognate Sxs. One hypothesis for SM protein function is that they facilitate a switch of the Sx from a closed to an open conformation, thus lifting the inhibitory action of the Habc domain and freeing the SNARE motif to participate in SNARE complexes. However, whether these regulatory mechanisms are conserved throughout the Sx/SM protein families remains contentious as it is not clear whether the closed conformation represents a universal feature of Sxs.

scholarly journals The Sec1/Munc18 protein Vps45 holds the Qa-SNARE Tlg2 in an open conformation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Travis J Eisemann ◽  
Frederick Allen ◽  
Kelly Lau ◽  
Gregory R Shimamura ◽  
Philip D Jeffrey ◽  
...  
Keyword(s):  
Snare Complex ◽  
Snare Proteins ◽  
Snare Protein ◽  
Sm Proteins ◽  
Closed Conformation ◽  
Open Conformation ◽  
Helical Hairpin ◽  
Snare Motif ◽  
Sm Protein ◽  
Template Complex

Fusion of intracellular trafficking vesicles is mediated by the assembly of SNARE proteins into membrane-bridging complexes. SNARE-mediated membrane fusion requires Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. Paradoxically, the SM protein Munc18-1 traps the Qa-SNARE protein syntaxin-1 in an autoinhibited closed conformation. Here we present the structure of a second SM–Qa-SNARE complex, Vps45–Tlg2. Strikingly, Vps45 holds Tlg2 in an open conformation, with its SNARE motif disengaged from its Habc domain and its linker region unfolded. The domain 3a helical hairpin of Vps45 is unfurled, exposing the presumptive R-SNARE binding site to allow template complex formation. Although Tlg2 has a pronounced tendency to form homo-tetramers, Vps45 can rescue Tlg2 tetramers into stoichiometric Vps45–Tlg2 complexes. Our findings demonstrate that SM proteins can engage Qa-SNAREs using at least two different modes, one in which the SNARE is closed and one in which it is open.

scholarly journals Mso1p Regulates Membrane Fusion through Interactions with the Putative N-Peptide–binding Area in Sec1p Domain 1

2010 ◽  
Vol 21 (8) ◽  
pp. 1362-1374 ◽  
Author(s):  
Marion Weber ◽  
Konstantin Chernov ◽  
Hilkka Turakainen ◽  
Gerd Wohlfahrt ◽  
Maria Pajunen ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Protein Interactions ◽  
Peptide Binding ◽  
Complex Function ◽  
Snare Complex ◽  
Targeted Mutagenesis ◽  
Rab Gtpase ◽  
Binding Modes ◽  
Sm Proteins ◽  
Sm Protein

Sec1p/Munc18 (SM) family proteins regulate SNARE complex function in membrane fusion through their interactions with syntaxins. In addition to syntaxins, only a few SM protein interacting proteins are known and typically, their binding modes with SM proteins are poorly characterized. We previously identified Mso1p as a Sec1p-binding protein and showed that it is involved in membrane fusion regulation. Here we demonstrate that Mso1p and Sec1p interact at sites of exocytosis and that the Mso1p–Sec1p interaction site depends on a functional Rab GTPase Sec4p and its GEF Sec2p. Random and targeted mutagenesis of Sec1p, followed by analysis of protein interactions, indicates that Mso1p interacts with Sec1p domain 1 and that this interaction is important for membrane fusion. In many SM family proteins, domain 1 binds to a N-terminal peptide of a syntaxin family protein. The Sec1p-interacting syntaxins Sso1p and Sso2p lack the N-terminal peptide. We show that the putative N-peptide binding area in Sec1p domain 1 is important for Mso1p binding, and that Mso1p can interact with Sso1p and Sso2p. Our results suggest that Mso1p mimics N-peptide binding to facilitate membrane fusion.

scholarly journals SNARE bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of membrane fusion

2010 ◽  
Vol 190 (1) ◽  
pp. 55-63 ◽  
Author(s):  
Jingshi Shen ◽  
Shailendra S. Rathore ◽  
Lavan Khandan ◽  
James E. Rothman
Keyword(s):  
Membrane Fusion ◽  
Complete Removal ◽  
Snare Complex ◽  
Sm Proteins ◽  
Membrane Bilayer ◽  
Core Domain ◽  
Target Membrane ◽  
Sm Protein ◽  
Typical Configuration ◽  
Intracellular Membrane Fusion

Sec1/Munc18 (SM) proteins activate intracellular membrane fusion through binding to cognate SNAP receptor (SNARE) complexes. The synaptic target membrane SNARE syntaxin 1 contains a highly conserved Habc domain, which connects an N-peptide motif to the SNARE core domain and is thought to participate in the binding of Munc18-1 (the neuronal SM protein) to the SNARE complex. Unexpectedly, we found that mutation or complete removal of the Habc domain had no effect on Munc18-1 stimulation of fusion. The central cavity region of Munc18-1 is required to stimulate fusion but not through its binding to the syntaxin Habc domain. SNAP-25, another synaptic SNARE subunit, contains a flexible linker and exhibits an atypical conjoined Qbc configuration. We found that neither the linker nor the Qbc configuration is necessary for Munc18-1 promotion of fusion. As a result, Munc18-1 activates a SNARE complex with the typical configuration, in which each of the SNARE core domains is individually rooted in the membrane bilayer. Thus, the SNARE four-helix bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of fusion.

scholarly journals Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association

2018 ◽  
Author(s):  
Junyi Jiao ◽  
Mengze He ◽  
Sarah A. Port ◽  
Richard W. Baker ◽  
Yonggang Xu ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Single Molecule ◽  
Force Spectroscopy ◽  
Snare Complex ◽  
Sm Proteins ◽  
Single Molecule Force Spectroscopy ◽  
Helix Bundle ◽  
The Stability ◽  
Sm Protein ◽  
Template Complex

AbstractSec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, but their mechanism(s) of action remain controversial. Using single-molecule force spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. Catalysis requires formation of an intermediate template complex in which Munc18-1 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while keeping their C-terminal regions separated. Next, SNAP-25 binds the templated SNAREs to form a partially-zippered SNARE complex. Finally, full zippering displaces Munc18-1. Munc18-1 mutations modulate the stability of the template complex in a manner consistent with their effects on membrane fusion, indicating that chaperoned SNARE assembly is essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a template complex, suggesting that SM protein mechanism is conserved.

scholarly journals Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

2004 ◽  
Vol 167 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Brenton L. Scott ◽  
Jeffrey S. Van Komen ◽  
Hassan Irshad ◽  
Song Liu ◽  
Kirilee A. Wilson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Fusion ◽  
Membrane Trafficking ◽  
Snare Complex ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Bud Neck ◽  
Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

scholarly journals Hemophagocytic lymphohistiocytosis caused by dominant-negative mutations in STXBP2 that inhibit SNARE-mediated membrane fusion

Blood ◽  
2015 ◽  
Vol 125 (10) ◽  
pp. 1566-1577 ◽  
Author(s):  
Waldo A. Spessott ◽  
Maria L. Sanmillan ◽  
Margaret E. McCormick ◽  
Nishant Patel ◽  
Joyce Villanueva ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Hemophagocytic Lymphohistiocytosis ◽  
Dominant Negative ◽  
Snare Complex ◽  
Complex Assembly ◽  
Mutant Proteins ◽  
Key Points ◽  
Lymphocyte Cytotoxicity ◽  
Dominant Negative Mutations

Key Points Monoallelic STXBP2 mutations affecting codon 65 impair lymphocyte cytotoxicity and contribute to hemophagocytic lymphohistiocytosis. Munc18-2R65Q/W mutant proteins function in a dominant-negative manner to impair membrane fusion and arrest SNARE-complex assembly.

scholarly journals Sec17 (α-SNAP) and Sec18 (NSF) restrict membrane fusion to R-SNAREs, Q-SNAREs, and SM proteins from identical compartments

2019 ◽  
Vol 116 (47) ◽  
pp. 23573-23581
Author(s):  
Youngsoo Jun ◽  
William Wickner
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Protein Complex ◽  
Negative Regulation ◽  
Rab Gtpases ◽  
Sm Proteins ◽  
Rab Family ◽  
Sm Protein

Membrane fusion at each organelle requires conserved proteins: Rab-GTPases, effector tethering complexes, Sec1/Munc18 (SM)-family SNARE chaperones, SNAREs of the R, Qa, Qb, and Qc families, and the Sec17/α-SNAP and ATP-dependent Sec18/NSF SNARE chaperone system. The basis of organelle-specific fusion, which is essential for accurate protein compartmentation, has been elusive. Rab family GTPases, SM proteins, and R- and Q-SNAREs may contribute to this specificity. We now report that the fusion supported by SNAREs alone is both inefficient and promiscuous with respect to organelle identity and to stimulation by SM family proteins or complexes. SNARE-only fusion is abolished by the disassembly chaperones Sec17 and Sec18. Efficient fusion in the presence of Sec17 and Sec18 requires a tripartite match between the organellar identities of the R-SNARE, the Q-SNAREs, and the SM protein or complex. The functions of Sec17 and Sec18 are not simply negative regulation; they stimulate fusion with either vacuolar SNAREs and their SM protein complex HOPS or endoplasmic reticulum/cis-Golgi SNAREs and their SM protein Sly1. The fusion complex of each organelle is assembled from its own functionally matching pieces to engage Sec17/Sec18 for fusion stimulation rather than inhibition.

scholarly journals The Sec1p/Munc18 protein Vps45p binds its cognate SNARE proteins via two distinct modes

2006 ◽  
Vol 173 (6) ◽  
pp. 927-936 ◽  
Author(s):  
Lindsay N. Carpp ◽  
Leonora F. Ciufo ◽  
Scott G. Shanks ◽  
Alan Boyd ◽  
Nia J. Bryant
Keyword(s):  
Crystal Structure ◽  
Membrane Trafficking ◽  
Protein Family ◽  
Snare Proteins ◽  
Outer Face ◽  
Sm Proteins ◽  
Hydrophobic Pocket ◽  
Second Mode ◽  
Sm Protein ◽  
Domain I

Sec1p/Munc18 (SM) proteins are essential for SNARE-mediated membrane trafficking. The formulation of unifying hypotheses for the function of the SM protein family has been hampered by the observation that two of its members bind their cognate syntaxins (Sxs) in strikingly different ways. The SM protein Vps45p binds its Sx Tlg2p in a manner analogous to that captured by the Sly1p–Sed5p crystal structure, whereby the NH2-terminal peptide of the Sx inserts into a hydrophobic pocket on the outer face of domain I of the SM protein. In this study, we report that although this mode of interaction is critical for the binding of Vps45p to Tlg2p, the SM protein also binds Tlg2p-containing SNARE complexes via a second mode that involves neither the NH2 terminus of Tlg2p nor the region of Vps45p that facilitates this interaction. Our findings point to the possibility that SM proteins interact with their cognate SNARE proteins through distinct mechanisms at different stages in the SNARE assembly/disassembly cycle.

scholarly journals A new role for RINT-1 in SNARE complex assembly at the trans-Golgi network in coordination with the COG complex

2013 ◽  
Vol 24 (18) ◽  
pp. 2907-2917 ◽  
Author(s):  
Kohei Arasaki ◽  
Daichi Takagi ◽  
Akiko Furuno ◽  
Miwa Sohda ◽  
Yoshio Misumi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Trafficking ◽  
Retrograde Transport ◽  
Snare Complex ◽  
Initial Contact ◽  
Complex Assembly ◽  
Trans Golgi Network ◽  
Cog Complex ◽  
Protein Receptor ◽  
Golgi Network

Docking and fusion of transport vesicles/carriers with the target membrane involve a tethering factor–mediated initial contact followed by soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE)–catalyzed membrane fusion. The multisubunit tethering CATCHR family complexes (Dsl1, COG, exocyst, and GARP complexes) share very low sequence homology among subunits despite likely evolving from a common ancestor and participate in fundamentally different membrane trafficking pathways. Yeast Tip20, as a subunit of the Dsl1 complex, has been implicated in retrograde transport from the Golgi apparatus to the endoplasmic reticulum. Our previous study showed that RINT-1, the mammalian counterpart of yeast Tip20, mediates the association of ZW10 (mammalian Dsl1) with endoplasmic reticulum–localized SNARE proteins. In the present study, we show that RINT-1 is also required for endosome-to–trans-Golgi network trafficking. RINT-1 uncomplexed with ZW10 interacts with the COG complex, another member of the CATCHR family complex, and regulates SNARE complex assembly at the trans-Golgi network. This additional role for RINT-1 may in part reflect adaptation to the demand for more diverse transport routes from endosomes to the trans-Golgi network in mammals compared with those in a unicellular organism, yeast. The present findings highlight a new role of RINT-1 in coordination with the COG complex.

Binding of UNC-18 to the N-terminus of syntaxin is essential for neurotransmission in Caenorhabditis elegans

2009 ◽  
Vol 418 (1) ◽  
pp. 73-80 ◽  
Author(s):  
James R. Johnson ◽  
Pawel Ferdek ◽  
Lu-Yun Lian ◽  
Jeff W. Barclay ◽  
Robert D. Burgoyne ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Membrane Fusion ◽  
Snare Complex ◽  
Binding Modes ◽  
Sm Proteins ◽  
Closed Conformation ◽  
Physiological Relevance ◽  
N Terminus

SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors) are widely accepted to drive all intracellular membrane fusion events. SM (Sec1/Munc18-like) proteins bind to SNAREs and this interaction may underlie their ubiquitous requirement for efficient membrane fusion. SM proteins bind to SNAREs in at least three modes: (i) to a closed conformation of syntaxin; (ii) to the syntaxin N-terminus; and (iii) to the assembled SNARE complex. Munc18-1 exhibits all three binding modes and recent in vitro reconstitution assays suggest that its interaction with the syntaxin N-terminus is essential for neuronal SNARE complex binding and efficient membrane fusion. To investigate the physiological relevance of these binding modes, we studied the UNC-18/UNC-64 SM/SNARE pair, which is essential for neuronal exocytosis in Caenorhabditis elegans. Mutations in the N-terminus of UNC-64 strongly inhibited binding to UNC-18, as did mutations targeting closed conformation binding. Complementary mutations in UNC-18 designed to selectively impair binding to either closed syntaxin or its N-terminus produced a similarly strong inhibition of UNC-64 binding. Therefore high-affinity UNC18/UNC-64 interaction in vitro involves both binding modes. To determine the physiological relevance of each mode, unc-18-null mutant worms were transformed with wild-type or mutant unc-18 constructs. The UNC-18(R39C) construct, that is defective in closed syntaxin binding, fully rescued the locomotion defects of the unc-18 mutant. In contrast, the UNC-18(F113R) construct, that is defective in binding to the N-terminus of UNC-64, provided no rescue. These results suggest that binding of UNC-18 to closed syntaxin is dispensable for membrane fusion, whereas interaction with the syntaxin N-terminus is essential for neuronal exocytosis in vivo.

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