Abstract
Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion in the secretory pathway. They contain conserved regions, termed SNARE motifs, that assemble between opposing membranes directionally from their N termini to their membrane-proximal C termini in a highly exergonic reaction. However, how this energy is utilized to overcome the energy barriers along the fusion pathway is still under debate. Here, we have used mutants of the SNARE synaptobrevin to arrest trans-SNARE zippering at defined stages. We have uncovered two distinct vesicle docking intermediates where the membranes are loosely and tightly connected, respectively. The tightly connected state is irreversible and independent of maintaining assembled SNARE complexes. Together, our results shed new light on the intermediate stages along the pathway of membrane fusion.
Highlights
Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion in the secretory pathway
Intracellular membrane fusion reactions in the eukaryotic secretory pathway are mostly catalyzed by soluble Nethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins (1–3)
We used an assay based on Förster resonance energy transfer (FRET) between two labeled SNAREs as described earlier (25)
Summary
NSF disassembles trans-SNARE complexes arrested in an almost completely zippered configuration. Addition of labeled syb(1–96) to liposomes containing the labeled acceptor complex resulted in a rapid decrease of the donor fluorescence (used here as a measure for FRET) This was reverted by the addition of NSF and ␣-SNAP in the presence of ATP (Fig. 1a). Very similar results were obtained when the full length was used rather than the N-terminally truncated variant of syntaxin (used here to generate a reactive SNARE acceptor complex (26)) (Fig. S1, A and B) These data show that NSF and ␣-SNAP disassemble trans complexes between tightly docked liposomes, we note here that syb⌬84 fluorescence did not recover fully to its initial levels (Fig. 1b).
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