scholarly journals HOPS catalyzes the interdependent assembly of each vacuolar SNARE into a SNARE complex

2017 ◽  
Vol 28 (7) ◽  
pp. 975-983 ◽  
Author(s):  
Amy Orr ◽  
Hongki Song ◽  
Scott F. Rusin ◽  
Arminja N. Kettenbach ◽  
William Wickner
Keyword(s):  
Lipid Membranes ◽  
Snare Complex ◽  
The Other ◽  
Rab Gtpases ◽  
Intracellular Membrane ◽  
Complex Assembly ◽  
Direct Binding ◽  
Sm Protein ◽  
Intracellular Membrane Fusion ◽  
Optimal Assembly

Rab GTPases, their effectors, SNAREs of the R, Qa, Qb, and Qc families, and SM SNARE-binding proteins catalyze intracellular membrane fusion. At the vacuole/lysosome, they are integrated by the homotypic fusion and vacuole protein sorting (HOPS) complex. Two HOPS subunits bind vacuolar Rabs for tethering, another binds the Qc SNARE, and a fourth HOPS subunit, an SM protein, has conserved grooves that bind R- and Qa-SNARE domains. Spontaneous quaternary SNARE complex assembly is very slow. We report an assay of SNARE complex assembly that does not rely on fusion and for which tethering does not coenrich the four SNAREs. HOPS is required in this assay for rapid SNARE complex assembly. Optimal assembly needs HOPS, lipid membranes to which the R- or Qa-SNARE and Ypt7:GTP are integrally bound, and each of the other three SNAREs. Each SNARE assembles into this complex relying on the others, suggesting four-SNARE complex assembly rather than direct binding of each to HOPS. SNAREs can be disassociated by Sec 17/Sec 18/ATP, completing a catalyzed cycle of SNARE assembly and disassembly.

scholarly journals Phosphatidylinositol and phosphatidylinositol-3-phosphate activate HOPS to catalyze SNARE assembly, allowing small headgroup lipids to support the terminal steps of membrane fusion

2021 ◽  
pp. mbc.E21-04-0191
Author(s):  
Thomas Torng ◽  
William Wickner
Keyword(s):  
Phosphatidic Acid ◽  
Membrane Fusion ◽  
Snare Complex ◽  
Rab Gtpases ◽  
Intracellular Membrane ◽  
Complex Assembly ◽  
Membrane Tethering ◽  
Intracellular Membrane Fusion ◽  
Phosphatidylinositol 3 ◽  
Hops Complex

Intracellular membrane fusion requires Rab GTPases, tethers, SNAREs of the R, Qa, Qb, and Qc families, and SNARE chaperones of the Sec17 (SNAP), Sec18 (NSF), and SM (Sec1/Munc18) families. The vacuolar HOPS complex combines the functions of membrane tethering and SM catalysis of SNARE assembly. HOPS is activated for this catalysis by binding to the vacuolar lipids and Rab. Of the 8 major vacuolar lipids, we now report that phosphatidylinositol and phosphatidylinositol-3-phosphate are required to activate HOPS for SNARE complex assembly. These lipids plus ergosterol also allow full trans-SNARE complex assembly, yet do not support fusion, which is reliant on either phosphatidylethanolamine (PE) or on phosphatidic acid (PA), phosphatidylserine (PS), and diacylglycerol (DAG). Fusion with a synthetic tether and without HOPS, or even without SNAREs, still relies on either PE or on PS, PA, and DAG. These lipids are thus required for the terminal bilayer rearrangement step of fusion, distinct from the lipid requirements for the earlier step of activating HOPS for trans-SNARE assembly.

scholarly journals Sec1p Binds to SNARE Complexes and Concentrates at Sites of Secretion

1999 ◽  
Vol 146 (2) ◽  
pp. 333-344 ◽  
Author(s):  
Chavela M. Carr ◽  
Eric Grote ◽  
Mary Munson ◽  
Frederick M. Hughson ◽  
Peter J. Novick
Keyword(s):  
Fluorescent Protein ◽  
Snare Complex ◽  
The Other ◽  
Complex Assembly ◽  
Vesicle Docking ◽  
Neuronal Protein ◽  
The Core ◽  
Target Membrane ◽  
Green Fluorescent ◽  
And Function

Proteins of the Sec1 family have been shown to interact with target-membrane t-SNAREs that are homologous to the neuronal protein syntaxin. We demonstrate that yeast Sec1p coprecipitates not only the syntaxin homologue Ssop, but also the other two exocytic SNAREs (Sec9p and Sncp) in amounts and in proportions characteristic of SNARE complexes in yeast lysates. The interaction between Sec1p and Ssop is limited by the abundance of SNARE complexes present in sec mutants that are defective in either SNARE complex assembly or disassembly. Furthermore, the localization of green fluorescent protein (GFP)-tagged Sec1p coincides with sites of vesicle docking and fusion where SNARE complexes are believed to assemble and function. The proposal that SNARE complexes act as receptors for Sec1p is supported by the mislocalization of GFP-Sec1p in a mutant defective for SNARE complex assembly and by the robust localization of GFP-Sec1p in a mutant that fails to disassemble SNARE complexes. The results presented here place yeast Sec1p at the core of the exocytic fusion machinery, bound to SNARE complexes and localized to sites of secretion.

scholarly journals Munc13 binds and recruits SNAP25 to chaperone SNARE complex assembly

2020 ◽  
Author(s):  
R Venkat Kalyana Sundaram ◽  
Huaizhou Jin ◽  
Feng Li ◽  
Tong Shu ◽  
Jeff Coleman ◽  
...  
Keyword(s):  
Single Molecule ◽  
Optical Tweezer ◽  
Snare Complex ◽  
Snare Proteins ◽  
Synaptic Membrane ◽  
Complex Assembly ◽  
Chaperone Function ◽  
Direct Binding ◽  
Synaptic Vesicle Fusion ◽  
Template Complex

ABSTRACTSynaptic vesicle fusion is mediated by membrane-bridging complexes formed by SNARE proteins - VAMP2 on the vesicle and Syntaxin-1/SNAP25 on the pre-synaptic membrane. Accumulating evidence suggest that chaperones Munc18-1 and Munc13-1 co-operatively catalyze SNARE assembly via an intermediate ‘template’ complex containing Syntaxin-1 and VAMP2. How SNAP25 is chaperoned into this nascent complex remains a mystery. Here we report that Munc13-1 recruits SNAP25 to initiate the ternary SNARE complex assembly by direct binding, as judged by bulk FRET spectroscopy and single-molecule optical tweezer studies. Detailed structure-function analyses show that the binding is mediated by the Munc13-1 MUN domain and is specific for the SNAP25 ‘linker’ region that connects the two SNARE motifs. Consequently, freely diffusing SNAP25 molecules on phospholipid bilayers are concentrated and presumably bound in ~1:1 stoichiometry by the self-assembled Munc13-1 nanoclusters. Our data suggests that Munc13-1’s capacity to bind all three synaptic SNARE proteins likely underlie its chaperone function.

scholarly journals Topological arrangement of the intracellular membrane fusion machinery

2011 ◽  
Vol 22 (14) ◽  
pp. 2612-2619 ◽  
Author(s):  
Shailendra S. Rathore ◽  
Nilanjan Ghosh ◽  
Yan Ouyang ◽  
Jingshi Shen
Keyword(s):  
Membrane Fusion ◽  
Fusion Reaction ◽  
Coiled Coil ◽  
Intracellular Membrane ◽  
Sm Proteins ◽  
Protein Receptors ◽  
Reconstituted System ◽  
Sm Protein ◽  
First Time ◽  
Intracellular Membrane Fusion

Soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) form a four-helix coiled-coil bundle that juxtaposes two bilayers and drives a basal level of membrane fusion. The Sec1/Munc18 (SM) protein binds to its cognate SNARE bundle and accelerates the basal fusion reaction. The question of how the topological arrangement of the SNARE helices affects the reactivity of the fusion proteins remains unanswered. Here we address the problem for the first time in a reconstituted system containing both SNAREs and SM proteins. We find that to be fusogenic a SNARE topology must support both basal fusion and SM stimulation. Certain topological combinations of exocytic SNAREs result in basal fusion but cannot support SM stimulation, whereas other topologies support SM stimulation without inducing basal fusion. It is striking that of all the possible topological combinations of exocytic SNARE helices, only one induces efficient fusion. Our results suggest that the intracellular membrane fusion complex is designed to fuse bilayers according to one genetically programmed topology.

scholarly journals Golgi SM protein Sly1 promotes productive trans-SNARE complex assembly through multiple mechanisms

2020 ◽  
Author(s):  
M. Duan ◽  
G. Gao ◽  
D.K. Banfield ◽  
A.J. Merz
Keyword(s):  
Snare Complex ◽  
Complex Assembly ◽  
Present Evidence ◽  
Closed Conformation ◽  
Close Range ◽  
Preferential Nucleation ◽  
Regulatory Loop ◽  
Sm Protein

SUMMARYSNARE chaperones of the Sec1/mammalian Unc-18 (SM) family have critical roles in SNARE-mediated membrane fusion. Using SNARE and Sly1 mutants, and a new in vitro assay of fusion, we separate and assess proposed mechanisms through which Sly1 augments fusion: (i) opening the closed conformation of the Qa-SNARE Sed5; (ii) close-range tethering of vesicles to target organelles, mediated by the Sly1-specific regulatory loop; and (iii) preferential nucleation of productive trans-SNARE complexes. We show that all three mechanisms are important and operate in parallel, and we present evidence that close-range tethering is particularly important for trans-complex assembly when cis-SNARE assembly is a competing process. In addition, the autoinhibitory N-terminal Habc domain of Sed5 has at least two positive activities: the Habc domain is needed for correct Sed5 localization, and it directly promotes Sly1-dependent fusion. Remarkably, “split Sed5,” with the Habc domain present only as a soluble fragment, is functional both in vitro and 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 SNARE zippering

2016 ◽  
Vol 36 (3) ◽  
Author(s):  
Xiaochu Lou ◽  
Yeon-Kyun Shin
Keyword(s):  
Recent Progress ◽  
Snare Complex ◽  
Intracellular Membrane ◽  
Attachment Protein ◽  
Receptor Proteins ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Associated Proteins ◽  
Intracellular Membrane Fusion ◽  
Factor Attachment Protein Receptor

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly conserved set of membrane-associated proteins that mediate intracellular membrane fusion. Cognate SNAREs from two separate membranes zipper to facilitate membrane apposition and fusion. Though the stable post-fusion conformation of SNARE complex has been extensively studied with biochemical and biophysical means, the pathway of SNARE zippering has been elusive. In this review, we describe some recent progress in understanding the pathway of SNARE zippering. We particularly focus on the half-zippered intermediate, which is most likely to serve as the main point of regulation by the auxiliary factors.

scholarly journals Complexin suppresses spontaneous exocytosis by capturing the membrane-proximal regions of VAMP2 and SNAP25

2019 ◽  
Author(s):  
J. Malsam ◽  
S. Bärfuss ◽  
T. Trimbuch ◽  
F. Zarebidaki ◽  
A.F.-P. Sonnen ◽  
...  
Keyword(s):  
Snare Complex ◽  
Intracellular Membrane ◽  
Attachment Protein ◽  
Neuronal Protein ◽  
Synaptic Release ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Living Neurons ◽  
Multiple Domains ◽  
Intracellular Membrane Fusion

SummaryThe neuronal protein complexin contains multiple domains that exert both clamping and facilitatory functions to tune spontaneous and action potential triggered synaptic release. We address the clamping mechanism and show that the accessory helix of complexin arrests the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that forms the core machinery of intracellular membrane fusion. In a reconstituted fusion assay, site- and stage-specific photo-cross-linking reveals that prior to fusion the complexin accessory helix laterally binds the membrane-proximal C-terminal ends of SNAP25 and VAMP2. Corresponding complexin interface mutants selectively increase spontaneous release of neurotransmitter in living neurons, implying that the accessory helix suppresses final zippering/assembly of the SNARE four-helix bundle by restraining VAMP2 and SNAP25.

scholarly journals A short region upstream of the yeast vacuolar Qa-SNARE heptad-repeats promotes membrane fusion through enhanced SNARE complex assembly

2017 ◽  
Vol 28 (17) ◽  
pp. 2282-2289 ◽  
Author(s):  
Hongki Song ◽  
William Wickner
Keyword(s):  
Snare Complex ◽  
Heptad Repeat ◽  
Fusion Activity ◽  
Complex Assembly ◽  
Protein Receptor ◽  
Repeat Domain ◽  
High Concentrations ◽  
Heptad Repeats ◽  
Sm Protein ◽  
Very High

Whereas SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor) heptad-repeats are well studied, SNAREs also have upstream N-domains of indeterminate function. The assembly of yeast vacuolar SNAREs into complexes for fusion can be studied in chemically defined reactions. Complementary proteoliposomes bearing a Rab:GTP and either the vacuolar R-SNARE or one of the three integrally anchored Q-SNAREs were incubated with the tethering/SM protein complex HOPS and the two other soluble SNAREs (lacking a transmembrane anchor) or their SNARE heptad-repeat domains. Fusion required a transmembrane-anchored R-SNARE on one membrane and an anchored Q-SNARE on the other. The N-domain of the Qb-SNARE was completely dispensable for fusion. Whereas fusion can be promoted by very high concentrations of the Qa-SNARE heptad-repeat domain alone, at physiological concentrations the Qa-SNARE heptad-repeat domain alone has almost no fusion activity. The 181–198 region of Qa, immediately upstream of the SNARE heptad-repeat domain, is required for normal fusion activity with HOPS. This region is needed for normal SNARE complex assembly.

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.

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