Distinct contributions of vacuolar Qabc- and R-SNARE proteins to membrane fusion specificity.
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ABSTRACT:
SUBMITTER: Izawa R
PROVIDER: S-EPMC3270998 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex facilitates intracellular membrane fusion. A single SNARE complex is thought to be insufficient; multiple copies of SNARE complexes must work cooperatively. However, the mechanism by which such a higher-order SNARE protein structure is assembled is unknown. EPR and fluorescence analyses show that at least three copies of target-membrane SNARE proteins self-assemble through the interaction between the transmembrane domains (TMDs), and this multimeric structure serves as scaffolding for trans-SNARE assembly. SNARE core formation in solution induces oligomerization of the TMDs of vesicle-associated SNAREs in the apposing membrane, transiently forming a supramolecular protein structure spanning two membranes. This higher-order protein intermediate evolves, by involving lipid molecules, to the hemifusion state. Hemifusion is subsequently followed by distal leaflet mixing and formation of the cis-SNARE complex.
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Project description:Intracellular membrane fusion requires R-SNAREs and Q-SNAREs to assemble into a four-helical parallel coiled-coil, with their hydrophobic anchors spanning the two apposed membranes. Based on the fusion properties of chemically defined SNARE- proteoliposomes, it has been proposed that the assembly of this helical bundle transduces force through the entire bilayer via the transmembrane SNARE anchor domains to drive fusion. However, an R-SNARE, Nyv1p, with a genetically engineered lipid anchor that spans half of the bilayer suffices for the fusion of isolated vacuoles, although this organelle has other R-SNAREs. To demonstrate unequivocally the fusion activity of lipid-anchored Nyv1p, we reconstituted proteoliposomes with purified lipid-anchored Nyv1p as the only protein. When these proteoliposomes were incubated with those bearing cognate Q-SNAREs, there was trans-SNARE complex assembly but, in accord with prior studies of the neuronal SNAREs, little lipid mixing. However, the addition of physiological fusion accessory proteins (HOPS, Sec17p, and Sec18p) allows lipid-anchored Nyv1p to support fusion, suggesting that trans-SNARE complex function is not limited to force transduction across the bilayers through the transmembrane domains.
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