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.

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.

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 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 Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles

2004 ◽  
Vol 167 (6) ◽  
pp. 1087-1098 ◽  
Author(s):  
Rutilio A. Fratti ◽  
Youngsoo Jun ◽  
Alexey J. Merz ◽  
Nathan Margolis ◽  
William Wickner
Keyword(s):  
Membrane Fusion ◽  
Protein Interactions ◽  
Fusion Proteins ◽  
Membrane Microdomains ◽  
Rab Gtpase ◽  
Protein Protein Interactions ◽  
Px Domain ◽  
Protein Partitioning ◽  
Membrane Fusion Proteins ◽  
Rab Effector

Membrane microdomains are assembled by lipid partitioning (e.g., rafts) or by protein–protein interactions (e.g., coated vesicles). During docking, yeast vacuoles assemble “vertex” ring-shaped microdomains around the periphery of their apposed membranes. Vertices are selectively enriched in the Rab GTPase Ypt7p, the homotypic fusion and vacuole protein sorting complex (HOPS)–VpsC Rab effector complex, SNAREs, and actin. Membrane fusion initiates at vertex microdomains. We now find that the “regulatory lipids” ergosterol, diacylglycerol and 3- and 4-phosphoinositides accumulate at vertices in a mutually interdependent manner. Regulatory lipids are also required for the vertex enrichment of SNAREs, Ypt7p, and HOPS. Conversely, SNAREs and actin regulate phosphatidylinositol 3-phosphate vertex enrichment. Though the PX domain of the SNARE Vam7p has direct affinity for only 3-phosphoinositides, all the regulatory lipids which are needed for vertex assembly affect Vam7p association with vacuoles. Thus, the assembly of the vacuole vertex ring microdomain arises from interdependent lipid and protein partitioning and binding rather than either lipid partitioning or protein interactions alone.

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 Characterization of two distinct binding modes between syntaxin 4 and Munc18c

2009 ◽  
Vol 419 (3) ◽  
pp. 655-660 ◽  
Author(s):  
Veronica Aran ◽  
Fiona M. Brandie ◽  
Alasdair R. Boyd ◽  
Theodoros Kantidakis ◽  
Elizabeth J. Rideout ◽  
...  
Keyword(s):  
Protein Function ◽  
Binding Mode ◽  
Binding Modes ◽  
Sm Proteins ◽  
Apparent Lack ◽  
Protein Receptor ◽  
Syntaxin 4 ◽  
Vacuolar Protein ◽  
Sm Protein

Interaction of SM (Sec1/Munc18) proteins with their cognate syntaxins represents an important regulatory mechanism of SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor)-mediated membrane fusion. Understanding the conserved mechanisms by which SM proteins function in this process has proved challenging, largely due to an apparent lack of conservation of binding mechanisms between different SM–syntaxin pairs. In the present study, we have identified a hitherto uncharacterized mode of binding between syntaxin 4 and Munc18c that is independent of the binding mode shown previously to utilize the N-terminal peptide of syntaxin 4. Our data demonstrate that syntaxin 4 and Munc18c interact via two distinct modes of binding, analogous to those employed by syntaxin 1a–Munc18a and syntaxin 16–Vps45p (vacuolar protein sorting 45). These data support the notion that all syntaxin/SM proteins bind using conserved mechanisms, and pave the way for the formulation of unifying hypotheses of SM protein function.

scholarly journals Structure-based Functional Analysis Reveals a Role for the SM Protein Sly1p in Retrograde Transport to the Endoplasmic Reticulum

2005 ◽  
Vol 16 (9) ◽  
pp. 3951-3962 ◽  
Author(s):  
Yujie Li ◽  
Dieter Gallwitz ◽  
Renwang Peng
Keyword(s):  
Endoplasmic Reticulum ◽  
Mutational Analysis ◽  
Retrograde Transport ◽  
Snare Complex ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Sm Proteins ◽  
Sm Protein

Sec1p/Munc18 (SM) proteins are essential for membrane fusion events in eukaryotic cells. Here we describe a systematic, structure-based mutational analysis of the yeast SM protein Sly1p, which was previously shown to function in anterograde endoplasmic reticulum (ER)-to-Golgi and intra-Golgi protein transport. Five new temperature-sensitive (ts) mutants, each carrying a single amino acid substitution in Sly1p, were identified. Unexpectedly, not all of the ts mutants exhibited striking anterograde ER-to-Golgi transport defects. For example, in cells of the novel sly1-5 mutant, transport of newly synthesized lysosomal and secreted proteins was still efficient, but the ER-resident Kar2p/BiP was missorted to the outside of the cell, and two proteins, Sed5p and Rer1p, which normally shuttle between the Golgi and the ER, failed to relocate to the ER. We also discovered that in vivo, Sly1p was associated with a SNARE complex formed on the ER, and that in vitro, the SM protein directly interacted with the ER-localized nonsyntaxin SNAREs Use1p/Slt1p and Sec20p. Furthermore, several conditional mutants defective in Golgi-to-ER transport were synthetically lethal with sly1-5. Together, these results indicate a previously unrecognized function of Sly1p in retrograde transport to the endoplasmic reticulum.

scholarly journals SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity

2017 ◽  
Vol 114 (11) ◽  
pp. E2176-E2185 ◽  
Author(s):  
Waldo A. Spessott ◽  
Maria L. Sanmillan ◽  
Margaret E. McCormick ◽  
Vineet V. Kulkarni ◽  
Claudio G. Giraudo
Keyword(s):  
Transmembrane Domain ◽  
Snare Complex ◽  
Complete Fusion ◽  
Sm Proteins ◽  
Granule Exocytosis ◽  
Cytolytic Granule ◽  
Syntaxin 11 ◽  
Sm Protein ◽  
Lytic Granule

The atypical lipid-anchored Syntaxin 11 (STX11) and its binding partner, the Sec/Munc (SM) protein Munc18-2, facilitate cytolytic granule release by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Patients carrying mutations in these genes develop familial hemophagocytic lymphohistiocytosis, a primary immunodeficiency characterized by impaired lytic granule exocytosis. However, whether a SNARE such as STX11, which lacks a transmembrane domain, can support membrane fusion in vivo is uncertain, as is the precise role of Munc18-2 during lytic granule exocytosis. Here, using a reconstituted “flipped” cell–cell fusion assay, we show that lipid-anchored STX11 and its cognate SNARE proteins mainly support exchange of lipids but not cytoplasmic content between cells, resembling hemifusion. Strikingly, complete fusion is stimulated by addition of wild-type Munc18-2 to the assay, but not of Munc18-2 mutants with abnormal STX11 binding. Our data reveal that Munc18-2 is not just a chaperone of STX11 but also directly contributes to complete membrane merging by promoting SNARE complex assembly. These results further support the concept that SM proteins in general are part of the core fusion machinery. This fusion mechanism likely contributes to other cell-type–specific exocytic processes such as platelet secretion.

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