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Distinct contributions of vacuolar Qabc- and R-SNARE proteins to membrane fusion specificity.

ABSTRACT: In eukaryotic endomembrane systems, Qabc-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) on one membrane and R-SNARE on the opposing membrane assemble into a trans-QabcR-SNARE complex to drive membrane fusion. However, it remains ambiguous whether pairing of Qabc- and R-SNAREs mediates membrane fusion specificity. Here, we explored the fusion specificity of reconstituted proteoliposomes bearing purified SNAREs in yeast vacuoles and other organelles. We found that not only vacuolar R-SNARE Nyv1p but also the non-cognate R-SNAREs, endosomal Snc2p, and endoplasmic reticulum-Golgi Sec22p caused efficient fusion with vacuolar Qabc-SNAREs. In contrast, their fusion is blocked completely by replacing vacuolar Qc-SNARE Vam7p with the non-cognate endosomal Tlg1p and Syn8p, although these endosomal Qc-SNAREs fully retained the ability to form cis-SNARE complexes with vacuolar SNAREs in solution and on membranes. Thus, our current study establishes that an appropriate assembly of Qabc-SNAREs is crucial for regulating fusion specificity, whereas R-SNARE itself has little contribution to specificity.

SUBMITTER: Izawa R

PROVIDER: S-EPMC3270998 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Distinct contributions of vacuolar Qabc- and R-SNARE proteins to membrane fusion specificity.

Izawa Ryota R Onoue Toshitaka T Furukawa Noriko N Mima Joji J

The Journal of biological chemistry 20111215 5

In eukaryotic endomembrane systems, Qabc-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) on one membrane and R-SNARE on the opposing membrane assemble into a trans-QabcR-SNARE complex to drive membrane fusion. However, it remains ambiguous whether pairing of Qabc- and R-SNAREs mediates membrane fusion specificity. Here, we explored the fusion specificity of reconstituted proteoliposomes bearing purified SNAREs in yeast vacuoles and other organelles. We found that ...[more]

PMID: 22174414

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Project description:Intracellular membrane traffic involves controlled membrane fission and fusion and is essential for eukaryotic cell homeostasis. Most intracellular fusion is facilitated by Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins, which catalyze membrane merging by assembly of a coiled helical bundle of four 60- to 70-residue "SNARE motifs." Perhaps no intracellular fusion reaction is as tightly regulated as that at the neuronal synapse, mediated by the synaptic vesicle SNARE Synaptobrevin-2 and the presynaptic plasma membrane SNAREs Syntaxin-1a and SNAP25. SNAP25 is different from its partner SNAREs: it contributes not one but two SNARE motifs to the final complex and instead of transmembrane domains is anchored in the membrane by post-translational palmitoylation of a long flexible linker between the SNARE motifs. Despite reports of structural and functional differences between the two SNARE motifs, many models of SNARE assembly and fusion consider SNAP25 to be a single functional unit and do not address how linking two distinct motifs in a single polypeptide contributes to synaptic SNARE assembly and fusion. To investigate whether SNAP25's two SNARE motifs regulate each other's folding and ability to assemble with other SNAREs, we determined their secondary structures in isolation and in the context of the whole protein by NMR spectroscopy and correlated the ability of the individual membrane-anchored SNARE motifs to interact with Syntaxin-1a and catalyze fusion in FRET-based binding and single-particle fusion assays, respectively. Our results demonstrate that the isolated N-terminal SNARE motif of SNAP25 promotes stronger Syntaxin-1a binding on membranes and more efficient fusion than wild-type SNAP25, while the C-terminal SNARE motif binds only transiently and facilitates kinetically delayed fusion. By comparing the functional properties of the single motifs to those of the full-length protein, we propose a new model of SNAP25 self-regulation in SNARE assembly and membrane fusion.

Dataset Information

Distinct contributions of vacuolar Qabc- and R-SNARE proteins to membrane fusion specificity.

Publications

Distinct contributions of vacuolar Qabc- and R-SNARE proteins to membrane fusion specificity.

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