scholarly journals HOPS Proofreads the trans-SNARE Complex for Yeast Vacuole Fusion

2008 ◽  
Vol 19 (6) ◽  
pp. 2500-2508 ◽  
Author(s):  
Vincent J. Starai ◽  
Christopher M. Hickey ◽  
William Wickner
Keyword(s):  
Snare Complex ◽  
Wild Type ◽  
Fusion Event ◽  
Sm Proteins ◽  
Terminal Domain ◽  
Domain Structures ◽  
Vacuole Fusion ◽  
Protein Receptors ◽  
Optimal Fusion ◽  
Guanosine Triphosphatase

The fusion of yeast vacuoles, like other organelles, requires a Rab-family guanosine triphosphatase (Ypt7p), a Rab effector and Sec1/Munc18 (SM) complex termed HOPS (homotypic fusion and vacuole protein sorting), and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The central 0-layer of the four bundled vacuolar SNAREs requires the wild-type three glutaminyl (Q) and one arginyl (R) residues for optimal fusion. Alterations of this layer dramatically increase the Km value for SNAREs to assemble trans-SNARE complexes and to fuse. We now find that added purified HOPS complex strongly suppresses the fusion of vacuoles bearing 0-layer alterations, but it has little effect on the fusion of vacuoles with wild-type SNAREs. HOPS proofreads at two levels, inhibiting the formation of trans-SNARE complexes with altered 0-layers and suppressing the ability of these mismatched 0-layer trans-SNARE complexes to support membrane fusion. HOPS proofreading also extends to other parts of the SNARE complex, because it suppresses the fusion of trans-SNARE complexes formed without the N-terminal Phox homology domain of Vam7p (Qc). Unlike some other SM proteins, HOPS proofreading does not require the Vam3p (Qa) N-terminal domain. HOPS thus proofreads SNARE domain and N-terminal domain structures and regulates the fusion capacity of trans-SNARE complexes, only allowing full function for wild-type SNARE configurations. This is the most direct evidence to date that HOPS is directly involved in the fusion event.

scholarly journals SM proteins Sly1 and Vps33 co-assemble with Sec17 and SNARE complexes to oppose SNARE disassembly by Sec18

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Braden T Lobingier ◽  
Daniel P Nickerson ◽  
Sheng-Ying Lo ◽  
Alexey J Merz
Keyword(s):  
Snare Complex ◽  
Wild Type ◽  
Sm Proteins ◽  
Working Model ◽  
Conformational Rearrangements ◽  
Kinetic Proofreading

Secretory and endolysosomal fusion events are driven by SNAREs and cofactors, including Sec17/α-SNAP, Sec18/NSF, and Sec1/Munc18 (SM) proteins. SMs are essential for fusion in vivo, but the basis of this requirement is enigmatic. We now report that, in addition to their established roles as fusion accelerators, SM proteins Sly1 and Vps33 directly shield SNARE complexes from Sec17- and Sec18-mediated disassembly. In vivo, wild-type Sly1 and Vps33 function are required to withstand overproduction of Sec17. In vitro, Sly1 and Vps33 impede SNARE complex disassembly by Sec18 and ATP. Unexpectedly, Sec17 directly promotes selective loading of Sly1 and Vps33 onto cognate SNARE complexes. A large thermodynamic barrier limits SM binding, implying that significant conformational rearrangements are involved. In a working model, Sec17 and SMs accelerate fusion mediated by cognate SNARE complexes and protect them from NSF-mediated disassembly, while mis-assembled or non-cognate SNARE complexes are eliminated through kinetic proofreading by Sec18.

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

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

ABSTRACTFusion of intracellular trafficking vesicles is mediated by the assembly of soluble N-ethylmaleimide-sensitive fusion protein receptors (SNAREs) to form membrane-bridging complexes. Also required for SNARE-mediated membrane fusion are Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. In the paradigmatic structure of an SM–SNARE complex, Munc18-1 bound to the Qa-SNARE syntaxin 1, the SNARE protein is trapped in an autoinhibited closed conformation that prevents it from entering into SNARE complexes. 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 three-helical 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. Tlg2 has a pronounced tendency to self-associate via its SNARE motif, and we demonstrate that Vps45 can rescue Tlg2 oligomers 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 Munc18a clusters SNARE-bearing liposomes prior to trans-SNARE zippering

2017 ◽  
Vol 474 (19) ◽  
pp. 3339-3354 ◽  
Author(s):  
Matthew Grant Arnold ◽  
Pratikshya Adhikari ◽  
Baobin Kang ◽  
Hao Xu (徐昊)
Keyword(s):  
Snare Complex ◽  
Binary Interaction ◽  
Fusion Reactions ◽  
Sm Proteins ◽  
Attachment Protein ◽  
Direct Role ◽  
Vesicle Docking ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Snare Complex Formation

Sec1–Munc18 (SM) proteins co-operate with SNAREs {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein] receptors} to mediate membrane fusion in eukaryotic cells. Studies of Munc18a/Munc18-1/Stxbp1 in neurotransmission suggest that SM proteins accelerate fusion kinetics primarily by activating the partially zippered trans-SNARE complex. However, accumulating evidence has argued for additional roles for SM proteins in earlier steps in the fusion cascade. Here, we investigate the function of Munc18a in reconstituted exocytic reactions mediated by neuronal and non-neuronal SNAREs. We show that Munc18a plays a direct role in promoting proteoliposome clustering, underlying vesicle docking during exocytosis. In the three different fusion reactions examined, Munc18a-dependent clustering requires an intact N-terminal peptide (N-peptide) motif in syntaxin that mediates the binary interaction between syntaxin and Munc18a. Importantly, clustering is preserved under inhibitory conditions that abolish both trans-SNARE complex formation and lipid mixing, indicating that Munc18a promotes membrane clustering in a step that is independent of trans-SNARE zippering and activation.

scholarly journals N-terminal domain of complexin independently activates calcium-triggered fusion

2016 ◽  
Vol 113 (32) ◽  
pp. E4698-E4707 ◽  
Author(s):  
Ying Lai ◽  
Ucheor B. Choi ◽  
Yunxiang Zhang ◽  
Minglei Zhao ◽  
Richard A. Pfuetzner ◽  
...  
Keyword(s):  
Membrane Binding ◽  
Snare Complex ◽  
Cooperative Interaction ◽  
Binding Property ◽  
Triggered Release ◽  
Terminal Domain ◽  
Influenza Virus Hemagglutinin ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Single Vesicle

Complexin activates Ca2+-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca2+-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca2+-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca2+-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca2+-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca2+-triggered release.

scholarly journals Stability, folding dynamics, and long-range conformational transition of the synaptic t-SNARE complex

2016 ◽  
Vol 113 (50) ◽  
pp. E8031-E8040 ◽  
Author(s):  
Xinming Zhang ◽  
Aleksander A. Rebane ◽  
Lu Ma ◽  
Feng Li ◽  
Junyi Jiao ◽  
...  
Keyword(s):  
Single Molecule ◽  
Plasma Membranes ◽  
Conformational Transition ◽  
Snare Complex ◽  
Structure Stability ◽  
Helix Bundle ◽  
Terminal Domain ◽  
C Terminus ◽  
Protein Receptors ◽  
Ionic Layer

Synaptic soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) couple their stepwise folding to fusion of synaptic vesicles with plasma membranes. In this process, three SNAREs assemble into a stable four-helix bundle. Arguably, the first and rate-limiting step of SNARE assembly is the formation of an activated binary target (t)-SNARE complex on the target plasma membrane, which then zippers with the vesicle (v)-SNARE on the vesicle to drive membrane fusion. However, the t-SNARE complex readily misfolds, and its structure, stability, and dynamics are elusive. Using single-molecule force spectroscopy, we modeled the synaptic t-SNARE complex as a parallel three-helix bundle with a small frayed C terminus. The helical bundle sequentially folded in an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a central ionic layer, with total unfolding energy of ∼17 kBT, where kB is the Boltzmann constant and T is 300 K. Peptide binding to the CTD activated the t-SNARE complex to initiate NTD zippering with the v-SNARE, a mechanism likely shared by the mammalian uncoordinated-18-1 protein (Munc18-1). The NTD zippering then dramatically stabilized the CTD, facilitating further SNARE zippering. The subtle bidirectional t-SNARE conformational switch was mediated by the ionic layer. Thus, the t-SNARE complex acted as a switch to enable fast and controlled SNARE zippering required for synaptic vesicle fusion and neurotransmission.

scholarly journals A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium

2007 ◽  
Vol 176 (3) ◽  
pp. 263-268 ◽  
Author(s):  
Adam C. Smith ◽  
Won Do Heo ◽  
Virginie Braun ◽  
Xiuju Jiang ◽  
Chloe Macrae ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Dominant Negative ◽  
Rab Gtpases ◽  
Wild Type ◽  
Intracellular Growth ◽  
Non Invasive ◽  
Serovar Typhimurium ◽  
Guanosine Triphosphatase ◽  
Kinetics Of

Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome–lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth.

scholarly journals Identification and Characterization of a Peptidoglycan Hydrolase, MurA, of Listeria monocytogenes, a Muramidase Needed for Cell Separation

2003 ◽  
Vol 185 (23) ◽  
pp. 6801-6808 ◽  
Author(s):  
Shannon A. Carroll ◽  
Torsten Hain ◽  
Ulrike Technow ◽  
Ayub Darji ◽  
Philippos Pashalidis ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Cell Separation ◽  
Bacterial Species ◽  
Surface Protein ◽  
Wild Type ◽  
Terminal Domain ◽  
Cell Wall Hydrolase ◽  
Characteristic Features ◽  
Type Gene

ABSTRACT A novel cell wall hydrolase encoded by the murA gene of Listeria monocytogenes is reported here. Mature MurA is a 66-kDa cell surface protein that is recognized by the well-characterized L. monocytogenes-specific monoclonal antibody EM-7G1. MurA displays two characteristic features: (i) an N-terminal domain with homology to muramidases from several gram-positive bacterial species and (ii) four copies of a cell wall-anchoring LysM repeat motif present within its C-terminal domain. Purified recombinant MurA produced in Escherichia coli was confirmed to be an authentic cell wall hydrolase with lytic properties toward cell wall preparations of Micrococcus lysodeikticus. An isogenic mutant with a deletion of murA that lacked the 66-kDa cell wall hydrolase grew as long chains during exponential growth. Complementation of the mutant strain by chromosomal reintegration of the wild-type gene restored expression of this murein hydrolase activity and cell separation levels to those of the wild-type strain. Studies reported herein suggest that the MurA protein is involved in generalized autolysis of L. monocytogenes.

scholarly journals Multiple mechanisms determine ER network morphology during the cell cycle in Xenopus egg extracts

2013 ◽  
Vol 203 (5) ◽  
pp. 801-814 ◽  
Author(s):  
Songyu Wang ◽  
Fabian B. Romano ◽  
Christine M. Field ◽  
Tim J. Mitchison ◽  
Tom A. Rapoport
Keyword(s):  
Cell Cycle ◽  
Adaptor Protein ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Egg Extracts ◽  
Organizing Center ◽  
Xenopus Egg ◽  
Guanosine Triphosphatase ◽  
Xenopus Egg Extracts ◽  
Network Morphology

In metazoans the endoplasmic reticulum (ER) changes during the cell cycle, with the nuclear envelope (NE) disassembling and reassembling during mitosis and the peripheral ER undergoing extensive remodeling. Here we address how ER morphology is generated during the cell cycle using crude and fractionated Xenopus laevis egg extracts. We show that in interphase the ER is concentrated at the microtubule (MT)-organizing center by dynein and is spread by outward extension of ER tubules through their association with plus ends of growing MTs. Fusion of membranes into an ER network is dependent on the guanosine triphosphatase atlastin (ATL). NE assembly requires fusion by both ATL and ER-soluble N-ethyl-maleimide–sensitive factor adaptor protein receptors. In mitotic extracts, the ER converts into a network of sheets connected by ER tubules and loses most of its interactions with MTs. Together, these results indicate that fusion of ER membranes by ATL and interaction of ER with growing MT ends and dynein cooperate to generate distinct ER morphologies during the cell cycle.

Abstract 1361: Leukocyte Antigen-Related Phosphatase Regulates Insulin Sensitivity by Interaction with Snapin

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Aleksandr E Vendrov ◽  
Igor Tchivilev ◽  
Xi-Lin Niu ◽  
Juxiang Li ◽  
Marschall S Runge ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Insulin Sensitivity ◽  
Glucose Uptake ◽  
Glycogen Synthase ◽  
Serine Phosphorylation ◽  
Snare Complex ◽  
Vascular Pathology ◽  
Wild Type ◽  
Membrane Translocation ◽  
Leukocyte Antigen

Several protein tyrosine phosphatases including leukocyte antigen-related (LAR) phosphatase have been implicated in insulin resistance, which is a risk factor for atherosclerosis. We showed previously that LAR negatively regulates insulin-like growth factor-1 (IGF1) signaling in vascular smooth muscle cells (VSMC) leading to increased proliferation and migration. Absence of LAR also enhanced neointima formation in response to arterial injury in mice. However, the role of LAR-modulated signaling in the development of insulin resistance has not been elucidated. Here, we investigated the function of LAR in regulating glucose uptake and insulin sensitivity. We identified snapin, a SNARE-associated protein involved in glucose transporter Glut4 vesicle fusion with plasma membrane, as a LAR-interacting protein using a yeast two-hybrid screen. IGF1-induced serine phosphorylation of snapin, its translocation to membrane and association with SNARE complex were enhanced in VSMC lacking LAR. Similarly, PI3K-PDK1-PKCζ signaling pathway was more active in LAR-/- cells after IGF1 treatment. This resulted in enhanced Glut4 activation, its membrane translocation and association with snapin. Glut4 membrane translocation and association with snapin after IGF1 treatment were impaired in snapin+/− VSMC. IGF1 treatment also increased serine phosphorylation of GSK3 β in LAR−/− VSMC leading to increased activation of glycogen synthase. Consistent with this, enhanced glucose uptake was observed in LAR−/− VSMC compared to wild-type cells after IGF1 treatment. Basal and IGF1-induced glucose uptake were significantly lower in snapin+/− VSMC than in wild-type cells. Snapin+/− mice had higher levels of blood glucose, lower quantitative insulin sensitivity check index (QUICKI) and impaired response to insulin in insulin tolerance test (ITT) compared to wild-type mice. Decrease of QUICKI and impairment of IIT were more pronounced in snapin+/− mice fed a high-fat diet. In addition, Doppler ultrasonography indicated increased arterial stiffness in snapin+/− mice. Together, these data indicate that LAR negatively regulates snapin phosphorylation which in turn affects glucose uptake leading to the development of insulin resistance and vascular pathology.

scholarly journals A Vacuolar v–t-SNARE Complex, the Predominant Form In Vivo and on Isolated Vacuoles, Is Disassembled and Activated for Docking and Fusion

1998 ◽  
Vol 140 (1) ◽  
pp. 61-69 ◽  
Author(s):  
Christian Ungermann ◽  
Benjamin J. Nichols ◽  
Hugh R.B. Pelham ◽  
William Wickner
Keyword(s):  
Atp Hydrolysis ◽  
Complex Structure ◽  
Snare Complex ◽  
Functional Aspect ◽  
Independent Function ◽  
Attachment Protein ◽  
Vacuole Fusion ◽  
Predominant Form ◽  
Target Membrane

Homotypic vacuole fusion in yeast requires Sec18p (N-ethylmaleimide–sensitive fusion protein [NSF]), Sec17p (soluble NSF attachment protein [α-SNAP]), and typical vesicle (v) and target membrane (t) SNAP receptors (SNAREs). We now report that vacuolar v- and t-SNAREs are mainly found with Sec17p as v–t-SNARE complexes in vivo and on purified vacuoles rather than only transiently forming such complexes during docking, and disrupting them upon fusion. In the priming reaction, Sec18p and ATP dissociate this v–t-SNARE complex, accompanied by the release of Sec17p. SNARE complex structure governs each functional aspect of priming, as the v-SNARE regulates the rate of Sec17p release and, in turn, Sec17p-dependent SNARE complex disassembly is required for independent function of the two SNAREs. Sec17p physically and functionally interacts largely with the t-SNARE. (a) Antibodies to the t-SNARE, but not the v-SNARE, block Sec17p release. (b) Sec17p is associated with the t-SNARE in the absence of v-SNARE, but is not bound to the v-SNARE without t-SNARE. (c) Vacuoles with t-SNARE but no v-SNARE still require Sec17p/Sec18p priming, whereas their fusion partners with v-SNARE but no t-SNARE do not. Sec18p thus acts, upon ATP hydrolysis, to disassemble the v–t-SNARE complex, prime the t-SNARE, and release the Sec17p to allow SNARE participation in docking and fusion. These studies suggest that the analogous ATP-dependent disassembly of the 20-S complex of NSF, α-SNAP, and v- and t-SNAREs, which has been studied in detergent extracts, corresponds to the priming of SNAREs for docking rather than to the fusion of docked membranes.

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