A Clamping Mechanism Involved in SNARE-Dependent Exocytosis

Giraudo, Claudio G.; Eng, William; Melia, Thomas J.; Rothman, James E.

doi:10.1126/science.1129450

Cited by 331 publications

(402 citation statements)

References 34 publications

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“…Current data suggest that SNARE complex formation proceeds in a vectorial manner from the N-terminal, membranedistal region toward the C-terminal, membrane-proximal end, which may draw the opposing membranes close enough together for fusion to proceed (Fiebig et al, 1999;Pobbati et al, 2006;Sorensen et al, 2006). Consistent with this, in reconstituted assay systems, SNAREs on their own can support membrane fusion (Weber et al, 1998;Hu et al, 2003;Giraudo et al, 2006;Pobbati et al, 2006).…”

Section: Introductionsupporting

confidence: 59%

A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

Fdez

Jowitt

Wang

et al. 2008

MBoC

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The interactions underlying the cooperativity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes during neurotransmission are not known. Here, we provide a molecular characterization of a dimer formed between the cytoplasmic portions of neuronal SNARE complexes. Dimerization generates a two-winged structure in which the C termini of cytosolic SNARE complexes are in apposition, and it involves residues from the vesicleassociated SNARE synaptobrevin 2 that lie close to the cytosol-membrane interface within the full-length protein.Mutation of these residues reduces stability of dimers formed between SNARE complexes, without affecting the stability of each individual SNARE complex. These mutations also cause a corresponding decrease in the ability of botulinum toxin-resistant synaptobrevin 2 to rescue regulated exocytosis in toxin-treated neuroendocrine cells. Moreover, such synaptobrevin 2 mutants give rise to a dominant-negative inhibition of exocytosis. These data are consistent with an important role for SNARE complex dimers in neurosecretion.

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Section: Introductionsupporting

confidence: 59%

A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

Fdez

Jowitt

Wang

et al. 2008

MBoC

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“…It has been well established that SNARE proteins function as engines for membrane fusion (Jahn and Scheller, 2006). The focus of the SNARE field has recently shifted to the regulation of SNARE functions (Giraudo et al, 2006). Another challenge in the field is to define the physiological role of individual SNAREs.…”

Section: Discussionmentioning

confidence: 99%

VAMP8/Endobrevin as a General Vesicular SNARE for Regulated Exocytosis of the Exocrine System

Wang

Shi

Guo

et al. 2007

MBoC

100

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The molecular mechanism governing the regulated secretion of most exocrine tissues remains elusive, although VAMP8/ endobrevin has recently been shown to be the major vesicular SNARE (v-SNARE) of zymogen granules of pancreatic exocrine acinar cells. In this article, we have characterized the role of VAMP8 in the entire exocrine system. Immunohistochemical studies showed that VAMP8 is expressed in all examined exocrine tissues such as salivary glands, lacrimal (tear) glands, sweat glands, sebaceous glands, mammary glands, and the prostate. Severe anomalies were observed in the salivary and lacrimal glands of VAMP8-null mice. Mutant salivary glands accumulated amylase and carbonic anhydrase VI. Electron microscopy revealed an accumulation of secretory granules in the acinar cells of mutant parotid and lacrimal glands. Pilocarpine-stimulated secretion of saliva proteins was compromised in the absence of VAMP8. Protein aggregates were observed in mutant lacrimal glands. VAMP8 may interact with syntaxin 4 and SNAP-23. These results suggest that VAMP8 may act as a v-SNARE for regulated secretion of the entire exocrine system.

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“…Complexin binds to SNARE complexes via its central α-helix, which inserts in an anti-parallel orientation into a groove formed by synaptobrevin/VAMP and syntaxin-1 [8,9]. Although multiple approaches have revealed an essential role of complexin in synaptic fusion [7,[10][11][12][13][14][15], the nature of this role remains unclear. In vertebrate autapses, deletion of complexin selectively impairs fast synchronous neurotransmitter release without changing asynchronous or spontaneous release [7,10].…”

mentioning

confidence: 99%

“…S4). Thus, the complexin knock-down greatly impaired fast synchronous but not asynchronous synaptic vesicle fusion, and increased spontaneous fusion, thereby reconciling the divergent phenotypes observed in vertebrate autapses, Drosophila neuromuscular junctions, and in vitro fusion assays [7,[10][11][12][13][14][15].We next examined which complexin sequences mediate spontaneous and evoked fusion (Fig. 1C).…”

mentioning

confidence: 99%

Complexin Controls the Force Transfer from SNARE Complexes to Membranes in Fusion

Maximov

Tang

Yang

et al. 2009

Science

315

489

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Trans-SNARE complexes catalyze fast synaptic vesicle fusion and bind complexin, but the function of complexin binding to SNARE complexes remains unclear. Here we show that in neuronal synapses, complexin simultaneously suppressed spontaneous fusion and activated fast Ca 2+ -evoked fusion. The dual function of complexin required SNARE binding, and additionally involved distinct N-terminal sequences of complexin that localize to the point where trans-SNARE complexes insert into the fusing membranes, suggesting that complexin controls the force that trans-SNARE complexes apply onto the fusing membranes. Consistent with this hypothesis, a mutation in the membrane insertion sequence of the v-SNARE synaptobrevin/VAMP phenocopied the complexin loss-of-function state without impairing complexin-binding to SNARE complexes. Thus, complexin probably activates and clamps the force-transfer from assembled trans-SNARE complexes onto fusing membranes.Synaptic vesicle fusion is driven by assembly of trans-SNARE complexes (or SNAREpins) from syntaxin-1 and SNAP-25 on the plasma membrane, and synaptobrevin/ VAMP on the vesicle membrane [1][2][3]. Ca 2+ then triggers fast synchronous synaptic vesicle fusion by binding to the Ca 2+ -sensor synaptotagmin [4][5][6]. Besides SNARE proteins and synaptotagmin, fast Ca 2+ -triggered fusion requires complexin [7]. Complexin is composed of short N-and C-terminal sequences and two central α-helices. Complexin binds to SNARE complexes via its central α-helix, which inserts in an anti-parallel orientation into a groove formed by synaptobrevin/VAMP and syntaxin-1 [8,9]. Although multiple approaches have revealed an essential role of complexin in synaptic fusion [7,[10][11][12][13][14][15], the nature of this role remains unclear. In vertebrate autapses, deletion of complexin selectively impairs fast synchronous neurotransmitter release without changing asynchronous or spontaneous release [7,10]. In in vitro fusion assays, conversely, addition of complexin causes a general block of SNARE-dependent fusion, indicating that complexin is a SNARE clamp [11][12][13][14]. In Drosophila neuromuscular synapses, deletion of complexin produces a >20-fold increase in spontaneous release but only a small decrease in evoked release [15]. Thus, the role of complexin in fusion is unclear. Moreover, even the importance of complexin SNARE- [16,17]. Here we addressed these questions with two complementary approaches -RNAi-dependent knockdown of complexin with rescue, and replacing wild-type synaptobrevin with specific mutants using synaptobrevin knockout (KO) mice [18].We knocked down complexin expression in cultured cortical neurons using an shRNA that targets both complexin-1 and -2, the only complexin isoforms significantly expressed in these neurons [19]. For this purpose, we used lentiviruses that simultaneously synthesize the complexin shRNA and either GFP, wild-type complexin-1, or 4M-mutant complexin-1 that is unable to bind to SNARE complexes (19,20). Lentivirus expressing GFP without the shRNA ...

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A Clamping Mechanism Involved in SNARE-Dependent Exocytosis

Cited by 331 publications

References 34 publications

A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

VAMP8/Endobrevin as a General Vesicular SNARE for Regulated Exocytosis of the Exocrine System

Complexin Controls the Force Transfer from SNARE Complexes to Membranes in Fusion

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