Inhibition of SNARE Complex Assembly Differentially Affects Kinetic Components of Exocytosis

Xu, Tao; Rammner, Burkhard; Margittai, Martin; Artalejo, Antonio R.; Neher, Erwin; Jahn, Reinhard

doi:10.1016/s0092-8674(00)81669-4

Cited by 292 publications

(264 citation statements)

References 56 publications

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“…However, other in situ experiments are consistent with SNAP-25 first interacting with VAMP before a strong interaction with syntaxin (Chen et al, 2001). Capacitance measurements of exocytosis in chromaffin cells are consistent with partially zippered SNARE complexes being a prelude to full zippering of the complex and exocytosis (Xu et al, 1999;Rettig and Neher, 2002).…”

Section: Introductionsupporting

confidence: 54%

The Structural and Functional Implications of Linked SNARE Motifs in SNAP25

Wang¹,

Bittner²,

Axelrod

et al. 2008

MBoC

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We investigated the functional and structural implications of SNAP25 having two SNARE motifs (SN1 and SN2). A membrane-bound, intramolecular FRET probe was constructed to report on the folding of N-terminal SN1 and C-terminal SN2 in living cells. Membrane-bound constructs containing either or both SNARE motifs were also singly labeled with donor or acceptor fluorophores. Interaction of probes with other SNAREs was monitored by the formation of SDSresistant complexes and by changes in FRET measured in vitro using spectroscopy and in the plasma membrane of living cells using TIRF microscopy. The probes formed the predicted SDS-resistant SNARE complexes. FRET measurements revealed that syntaxin induced a close association of the N-termini of SN1 and SN2. This association required that the SNARE motifs reside in the same molecule. Unexpectedly, the syntaxin-induced FRET was prevented by VAMP. Both full-length SNAP25 constructs and the combination of its separated, membrane-bound constituent chains supported secretion in permeabilized chromaffin cells that had been allowed to rundown. However, only full-length SNAP25 constructs enabled robust secretion from intact cells or permeabilized cells before rundown. The experiments suggest that the bidentate structure permits specific conformations in complexes with syntaxin and VAMP and facilitates the function of SN1 and SN2 in exocytosis.

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Section: Introductionsupporting

confidence: 54%

The Structural and Functional Implications of Linked SNARE Motifs in SNAP25

Wang¹,

Bittner²,

Axelrod

et al. 2008

MBoC

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“…5), which may indicate that vesicles entering the slow exocytosis are characterized by a loose form of the SNARE complex and those that undergo rapid exocytosis by a tight form of the SNARE complex (37). Xu et al (37) interpreted that the two pools of vesicles that share a similar Ca 2ϩ sensitivity, are coupled sequentially, although a parallel pathway of exocytosis could not be excluded experimentally. Interestingly, our studies of the Ca 2ϩ sensitivity of exocytosis (Fig.…”

Section: Discussionmentioning

confidence: 99%

Rapid regulated dense-core vesicle exocytosis requires the CAPS protein

Rupnik

Kreft

Sikdar

et al. 2000

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

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Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca 2؉ -dependent activator protein for secretion), a protein required for Ca 2؉ -dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. Flash photolysis of caged Ca 2؉ elicited biphasic capacitance increases consisting of rapid and slow components with distinct Ca 2؉ dependencies. A threshold of Ϸ10 M Ca 2؉ was required to trigger the slow component, while the rapid capacitance increase was recorded already at a intracellular Ca 2؉ activity < 10 M. Both kinetic membrane capacitance components were abolished by botulinum neurotoxin B or E treatment, suggesting involvement of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicle fusion. The rapid but not the slow component was inhibited by CAPS antibody. These results were further clarified by immunocytochemical studies that revealed that CAPS was present on only a subset of dense-core vesicles. Overall, the results indicate that dense-core vesicle exocytosis in melanotrophs occurs by two parallel pathways. The faster pathway exhibits high sensitivity to Ca 2؉ and requires the presence of CAPS, which appears to act at a late stage in the secretory pathway.capacitance ͉ pituitary cells ͉ rat melanotrophs

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“…This most likely indicates that it competes with VAMP2 for formation of trans-SNARE complexes in the rescue but not the regular assay, thus preventing membrane fusion. Because loose or incompletely zippered SNARE complexes are thought to form during the early MgATP priming step of exocytosis (24,25,63), this suggests that amisyn probably acts upstream of priming when the SNAREs are fully accessible, a state that is prolonged in toxintreated cells because one of the four coils is incomplete. This need not mean that amisyn acts as a competitor in vivo, because it is possible that the coil domain without the N terminus acts in a dominant negative fashion, similar to the inhibitory effect of transmembrane-less VAMP coil domain (with or without its N terminus) on fusion (18).…”

Section: Amisyn Coil Domain Inhibits the Rescue Of Exocytosis By Snapmentioning

confidence: 99%

Amisyn, a Novel Syntaxin-binding Protein That May Regulate SNARE Complex Assembly

Scales

Hesser

Masuda

et al. 2002

Journal of Biological Chemistry

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The regulation of SNARE complex assembly likely plays an important role in governing the specificity as well as the timing of membrane fusion. Here we identify a novel brain-enriched protein, amisyn, with a tomosynand VAMP-like coiled-coil-forming domain that binds specifically to syntaxin 1a and syntaxin 4 both in vitro and in vivo, as assessed by co-immunoprecipitation from rat brain. Amisyn is mostly cytosolic, but a fraction cosediments with membranes. The amisyn coil domain can form SNARE complexes of greater thermostability than can VAMP2 with syntaxin 1a and SNAP-25 in vitro, but it lacks a transmembrane anchor and so cannot act as a v-SNARE in this complex. The amisyn coil domain prevents the SNAP-25 C-terminally mediated rescue of botulinum neurotoxin E inhibition of norepinephrine exocytosis in permeabilized PC12 cells to a greater extent than it prevents the regular exocytosis of these vesicles. We propose that amisyn forms nonfusogenic complexes with syntaxin 1a and SNAP-25, holding them in a conformation ready for VAMP2 to replace it to mediate the membrane fusion event, thereby contributing to the regulation of SNARE complex formation.The exocytosis of synaptic and dense core vesicles with the plasma membrane in neurons and neuroendocrine cells requires proteins of the SNARE 1 families. The vesicle-associated membrane protein, VAMP2, a v-or R-SNARE, forms a specific SNARE complex with the target membrane-associated t-or Q-SNAREs syntaxin 1 and SNAP-25, whose parallel orientation brings the two membranes in close enough proximity to fuse (1, 2). Structurally the SNARE complex is a four-helix bundle comprised of one coiled-coil-forming domain from each of syntaxin and VAMP and two from 4). The center of the bundle is made up of 15 hydrophobic layers from the "a" and "d" positions of the heptad repeats of these coiled-coilforming domains, whereas the central "ionic" layer is highly conserved and polar in nature, containing a glutamine residue in the three t-SNAREs and an arginine in the v-SNARE (3), hence the classification of v-and t-SNAREs as R-and QSNAREs, respectively (5). The parallel orientation and high stability of the SNARE bundle led to the proposal that its formation drives the mixing of the membrane bilayers by zippering up through the transmembrane domains of syntaxin and VAMP (1, 2), and there is some evidence to support this (6 -9). After the fusion event, the SNAREs are in a cis-complex in the same membrane and must be dissociated by the action of the ATPase NSF and ␣-SNAP, so that SNAREs can be regenerated for the next round of fusion (10 -13).In addition to mediating the fusion event, the SNARE proteins have also been implicated in the specificity of membrane fusion, the SNARE hypothesis stating that a particular v-SNARE on a transport vesicle should only form a specific complex with its cognate t-SNARE on the correct target membrane, thereby ensuring that the vesicles only fuse with the right compartment (14). Although this hypothesis was cast into doubt by the finding that soluble ...

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Inhibition of SNARE Complex Assembly Differentially Affects Kinetic Components of Exocytosis

Cited by 292 publications

References 56 publications

The Structural and Functional Implications of Linked SNARE Motifs in SNAP25

The Structural and Functional Implications of Linked SNARE Motifs in SNAP25

Rapid regulated dense-core vesicle exocytosis requires the CAPS protein

Amisyn, a Novel Syntaxin-binding Protein That May Regulate SNARE Complex Assembly

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