Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p

Nicholson, Karin L.; Munson, Mary; Miller, Rebecca; Filip, Thomas J.; Fairman, Robert; Hughson, Frederick M.

doi:10.1038/1834

Cited by 211 publications

(276 citation statements)

References 54 publications

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“…A similar assembly rate of ϳ6000 M Ϫ1 s Ϫ1 was observed for the interaction of the core regions of the exocytotic yeast QSNAREs Sso1p (syntaxin homologue) and Sec9p (SNAP-25 homologue) (21). The yeast Q-SNARE complex consists of a 1:1 heterodimer (20, 21), forming a three-helix bundle that is structured only in the N-terminal two-thirds of the three participating SNARE motifs (22).…”

Section: Discussionmentioning

confidence: 88%

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A Transient N-terminal Interaction of SNAP-25 and Syntaxin Nucleates SNARE Assembly

Fasshauer

Margittai²

2004

Journal of Biological Chemistry

191

239

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The SNARE proteins syntaxin, SNAP-25, and synaptobrevin play a central role during Ca 2؉ -dependent exocytosis at the nerve terminal. Whereas syntaxin and SNAP-25 are located in the plasma membrane, synaptobrevin resides in the membrane of synaptic vesicles. It is thought that gradual assembly of these proteins into a membrane-bridging ternary SNARE complex ultimately leads to membrane fusion. According to this model, syntaxin and SNAP-25 constitute an acceptor complex for synaptobrevin. In vitro, however, syntaxin and SNAP-25 form a stable complex that contains two syntaxin molecules, one of which is occupying and possibly obstructing the binding site of synaptobrevin. To elucidate the assembly pathway of the synaptic SNAREs, we have now applied a combination of fluorescence and CD spectroscopy. We found that SNARE assembly begins with the slow and rate-limiting interaction of syntaxin and SNAP-25. Their interaction was prevented by N-terminal but not by C-terminal truncations, suggesting that for productive assembly all three participating helices must come together simultaneously. This suggests a complicated nucleation process that might be the reason for the observed slow assembly rate. N-terminal truncations of SNAP-25 and syntaxin also prevented the formation of the ternary complex, whereas neither Nnor C-terminal shortened synaptobrevin helices lost their ability to interact. This suggests that binding of synaptobrevin occurs after the establishment of the syntaxin-SNAP-25 interaction. Moreover, binding of synaptobrevin was inhibited by an excess of syntaxin, suggesting that a 1:1 interaction of syntaxin and SNAP-25 serves as the on-pathway SNARE assembly intermediate.Following a Ca 2ϩ influx into the synaptic terminal, neurotransmitter is rapidly released from synaptic vesicles that fuse with the plasma membrane. The synaptic vesicle protein synaptobrevin 2 (also referred to as vesicle-associated membrane protein 2, or VAMP 2) and the plasma membrane proteins syntaxin 1a and SNAP-25 1 play a central role during this process. They belong to the family of so-called soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, which are essential for all vesicular trafficking steps. It is believed that SNAREs generally initiate the process of membrane fusion by the sequential formation of a stable heteromeric "trans" complex between the vesicular and the target membranes. This basic SNARE machinery is thought to be tightly controlled by a complex protein network (for a review, see Refs. 1-5).Syntaxin and synaptobrevin each contain a single SNARE complex-forming coiled-coil region, termed the SNARE motif, directly adjacent to their C-terminal transmembrane domain. Syntaxin 1a contains an additional regulatory N-terminal region, called the Habc domain. SNAP-25 is composed of two SNARE motifs connected by a long linker and is attached to the membrane by palmitoyl modifications in this region. Upon SNARE complex formation, major conformational changes occur, with mostly unstructured pr...

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Section: Discussionmentioning

confidence: 88%

“…The core region of the yeast Q-SNARE complex assembles with a relatively slow rate of ϳ6000 M Ϫ1 s Ϫ1 (21). This rate of Q-SNARE assembly was found to be rate-limiting for the assembly of the ternary complex.…”

mentioning

confidence: 99%

A Transient N-terminal Interaction of SNAP-25 and Syntaxin Nucleates SNARE Assembly

Fasshauer

Margittai²

2004

Journal of Biological Chemistry

191

239

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“…Indirect evidence suggests that more SNAP-23 proteins were expressed on the cell surface than SNAP-25 proteins. Among the yeast SNARE proteins that mediate exocytosis, Sso1p (homologue of syntaxin1 and syntaxin4) and Sec9p (homologue of SNAP-23 and SNAP-25) form a 1:1 complex, which constitutes a threehelix bundle that serves as a binding site for Snc1/2p (homologue to VAMP2 and VAMP3) [36,37]. It is well established that Syntaxin1 and SNAP-25 form a stoichimetric complex [4].…”

Section: Expression Of Flipped Snare Proteins On the Cell Surfacementioning

confidence: 99%

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

Hardee

Minnear

2007

Experimental Cell Research

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Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of α-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the Nterminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the Nterminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins.

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“…Prenylated Ypt1p was expressed in yeast as a His 6 -tagged fusion protein and purified from the membrane fraction as described before (23). Sso1p (amino acids 1-265) and Sec9p (amino acids 416 -651) were purified as described before (24). His 6 -Ypt1p was pre-loaded with nucleotide as described previously (25).…”

Section: Methodsmentioning

confidence: 99%

The Yip1p·Yif1p Complex Is Required for the Fusion Competence of Endoplasmic Reticulum-derived Vesicles

Barrowman¹,

Wang²,

Zhang

et al. 2003

Journal of Biological Chemistry

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Here we report that Yip1p and Yif1p, two members of an integral membrane protein complex that bind to the Rab Ypt1p, are required for membrane fusion with the Golgi in vitro. To block fusion, anti-Yip1p or anti-Yif1p antibodies must be added before vesicles bud from the endoplasmic reticulum (ER). These antibodies do not block the packaging of Yip1p, Yif1p, or the soluble NSF attachment protein receptor (SNAREs) into vesicles. We propose that Yip1p and Yif1p perform a critical role in establishing the fusion competence of ER to Golgi vesicles at the time of budding. Consistent with this proposal, we observe that the Yip1p⅐Yif1p complex binds to the ER to Golgi SNAREs Bos1p and Sec22p, two components of the membrane fusion machinery.

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Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p

Cited by 211 publications

References 54 publications

A Transient N-terminal Interaction of SNAP-25 and Syntaxin Nucleates SNARE Assembly

A Transient N-terminal Interaction of SNAP-25 and Syntaxin Nucleates SNARE Assembly

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

The Yip1p·Yif1p Complex Is Required for the Fusion Competence of Endoplasmic Reticulum-derived Vesicles

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