Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations

Weninger, Keith; Bowen, Mark E.; Chu, Steven; Brunger, Axel T.

doi:10.1073/pnas.2036428100

Cited by 122 publications

(161 citation statements)

References 44 publications

Supporting

Mentioning

154

Contrasting

Order By: Relevance

“…Purified Cpx A8C, Cpx 1-83 E25C, and commercial BSA were labeled with Alexa 647-maleimide in the presence of 5 mM TCEP overnight, following the protocol described in ref. 62. The labeling efficiency was assessed by absorbance spectroscopy: it was ∼40% for both the Cpx A8C and Cpx 1-83 E25C.…”

Section: Methodsmentioning

confidence: 99%

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

Gong

Lai

et al. 2016

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

Self Cite

View full text Add to dashboard Cite

In presynaptic nerve terminals, complexin regulates spontaneous "mini" neurotransmitter release and activates Ca 2+ -triggered synchronized neurotransmitter release. We studied the role of the C-terminal domain of mammalian complexin in these processes using singleparticle optical imaging and electrophysiology. The C-terminal domain is important for regulating spontaneous release in neuronal cultures and suppressing Ca 2+ -independent fusion in vitro, but it is not essential for evoked release in neuronal cultures and in vitro. This domain interacts with membranes in a curvature-dependent fashion similar to a previous study with worm complexin [Snead D, Wragg RT, Dittman JS, Eliezer D (2014) Membrane curvature sensing by the C-terminal domain of complexin. Nat Commun 5:4955]. The curvature-sensing value of the C-terminal domain is comparable to that of α-synuclein. Upon replacement of the C-terminal domain with membrane-localizing elements, preferential localization to the synaptic vesicle membrane, but not to the plasma membrane, results in suppression of spontaneous release in neurons. Membrane localization had no measurable effect on evoked postsynaptic currents of AMPA-type glutamate receptors, but mislocalization to the plasma membrane increases both the variability and the mean of the synchronous decay time constant of NMDA-type glutamate receptor evoked postsynaptic currents.

show abstract

Section: Methodsmentioning

confidence: 99%

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

Gong

Lai

et al. 2016

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

Self Cite

View full text Add to dashboard Cite

show abstract

“…SNARE Complex Assembly-The ternary SNARE complex was assembled and purified as described previously (31). Briefly, SNARE complexes were formed by adding SNAP25 to syntaxin, followed by the addition of VAMP2 (35-37) in a ratio of 1:2:3 (syntaxin/SNAP25/VAMP2), generating SDSresistant SNARE complexes (38).…”

Section: Methodsmentioning

confidence: 99%

Vesicle-associated Membrane Protein (VAMP) Cleavage by a New Metalloprotease from the Brazilian Scorpion Tityus serrulatus

Fletcher

Weninger

et al. 2010

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

We present evidence that venom from the Brazilian scorpion Tityus serrulatus and a purified fraction selectively cleave essential SNARE proteins within exocrine pancreatic tissue. Western blotting for vesicle-associated membrane protein type v-SNARE proteins (or synaptobrevins) reveals characteristic alterations to venom-treated excised pancreatic lobules in vitro. Immunocytochemistry by electron microscopy confirms both the SNARE identity as VAMP2 and the proteolysis of VAMP2 as a marked decrease in secondary antibody-conjugated colloidal gold particles that are predominantly associated with mature zymogen granules. Studies with recombinant SNARE proteins were used to determine the specific cleavage site in VAMP2 and the susceptibility of VAMP8 (endobrevin). The VAMP2 cleavage site is between the transmembrane anchor and the SNARE motif that assembles into the ternary SNARE complex. Inclusion of divalent chelating agents (EDTA) with fraction , an otherwise active purified component from venom, eliminates SNARE proteolysis, suggesting the active protein is a metalloprotease. The unique cleavages of VAMP2 and VAMP8 may be linked to pancreatitis that develops following scorpion envenomation as both of these v-SNARE proteins are associated with zymogen granule membranes in pancreatic acinar cells. We have isolated antarease, a metalloprotease from fraction that cleaves VAMP2, and report its amino acid sequence.

show abstract

“…37). Because SNARE complexes can assemble in both parallel and antiparallel fashions (40), such a role could ensure the proper orientation and specificity in SNARE complex formation. However, a role for SM proteins in providing SNARE complex specificity is difficult to reconcile with the relatively small number of SM proteins in yeast (four) and vertebrates (seven).…”

Section: Function Of Munc18-1 and Other Sm Proteins In Fusionmentioning

confidence: 99%

Munc18-1 binds directly to the neuronal SNARE complex

Dulubova¹,

Khvotchev²,

Liu³

et al. 2007

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

308

365

View full text Add to dashboard Cite

Both SM proteins (for Sec1/Munc18-like proteins) and SNARE proteins (for soluble NSF-attachment protein receptors) are essential for intracellular membrane fusion, but the general mechanism of coupling between their functions is unclear, in part because diverse SM protein/SNARE binding modes have been described. During synaptic vesicle exocytosis, the SM protein Munc18-1 is known to bind tightly to the SNARE protein syntaxin-1, but only when syntaxin-1 is in a closed conformation that is incompatible with SNARE complex formation. We now show that Munc18-1 also binds tightly to assembled SNARE complexes containing syntaxin-1. The newly discovered Munc18-1/SNARE complex interaction involves contacts of Munc18-1 with the N-terminal H abc domain of syntaxin-1 and the four-helical bundle of the assembled SNARE complex. Together with earlier studies, our results suggest that binding of Munc18-1 to closed syntaxin-1 is a specialization that evolved to meet the strict regulatory requirements of neuronal exocytosis, whereas binding of Munc18-1 to assembled SNARE complexes reflects a general function of SM proteins involved in executing membrane fusion.exocytosis ͉ membrane fusion ͉ neurotransmitter release ͉ Sec1/Munc18-like proteins ͉ synapse E very eukaryotic cell relies on the precise and regulated trafficking of proteins, membranes, and other types of cargo between cellular compartments. The molecular machinery responsible for membrane traffic is partly conserved to provide for basic membrane fusion and fission reactions and is partly cell type-and compartment-specific to meet the unique requirements of a particular cellular locale (1). Neurons have arguably the most complex membrane trafficking of all cells, primarily because synaptic transmission requires continuous (but at the same time tightly regulated) synaptic membrane traffic. At a synapse, neurotransmitter release is effected by fusion of synaptic vesicles with the presynaptic plasma membrane. Synaptic vesicle fusion requires two conserved protein families that are universally involved in membrane fusion reactions: SNARE proteins, which are thought to pull membranes together by forming tight ''SNARE complexes,'' and Sec1/Munc18-like proteins (SM proteins), which perform an unknown but essential role in fusion and interact with SNARE proteins (reviewed in refs. 2-4).Synaptic vesicle exocytosis involves one SM protein (Munc18-1) and three SNARE proteins (synaptobrevin/VAMP on synaptic vesicles, and SNAP-25 and syntaxin-1A/1B on the plasma membrane; syntaxin-1A and -1B are highly homologous and thought to have identical functions). All SNARE proteins contain a conserved 60-to 70-residue sequence, the SNARE motif, that assembles into a four-helical bundle during SNARE complex formation (5). Synaptobrevin/VAMP and SNAP-25 are ''minimal SNARE proteins'' that are composed of one (synaptobrevin) or two (SNAP-25) SNARE motifs and a membraneanchor sequence. In syntaxin-1, however, the SNARE motif and transmembrane region occupy less than half of the sequence, with the...

show abstract

Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations

Cited by 122 publications

References 44 publications

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

Vesicle-associated Membrane Protein (VAMP) Cleavage by a New Metalloprotease from the Brazilian Scorpion Tityus serrulatus

Munc18-1 binds directly to the neuronal SNARE complex

Contact Info

Product

Resources

About