Close is not enough: SNARE-dependent membrane fusion requires an active mechanism that transduces force to membrane anchors.
Is membrane fusion an essentially passive or an active process? It could be that fusion proteins simply need to pin two bilayers together long enough, and the bilayers could do the rest spontaneously. Or, it could be that the fusion proteins play an active role after pinning two bilayers, exerting force in the bilayer in one or another way to direct the fusion process. To distinguish these alternatives, we replaced one or both of the peptidic membrane anchors of exocytic vesicle (v)- and target membrane (t)-SNAREs (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor) with covalently attached lipids. Replacing either anchor with a phospholipid prevented fusion of liposomes by the isolated SNAREs, but still allowed assembly of trans-SNARE complexes docking vesicles. This result implies an active mechanism; if fusion occurred passively, simply holding the bilayers together long enough would have been sufficient. Studies using polyisoprenoid anchors ranging from 15-55 carbons and multiple phospholipid-containing anchors reveal distinct requirements for anchors of v- and t-SNAREs to function: v-SNAREs require anchors capable of spanning both leaflets, whereas t-SNAREs do not, so long as the anchor is sufficiently hydrophobic. These data, together with previous results showing fusion is inhibited as the length of the linker connecting the helical bundle-containing rod of the SNARE complex to the anchors is increased (McNew, J.A., T. Weber, D.M. Engelman, T.H. Sollner, and J.E. Rothman, 1999. Mol. Cell. 4:415-421), suggests a model in which one activity of the SNARE complex promoting fusion is to exert force on the anchors by pulling on the linkers. This motion would lead to the simultaneous inward movement of lipids from both bilayers, and in the case of the v-SNARE, from both leaflets.
McNew
J A
JA
Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Weber
T
T
Parlati
F
F
Johnston
R J
RJ
Melia
T J
TJ
Söllner
T H
TH
Rothman
J E
JE
D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D008561:Membrane Fusion / Q000502:physiology*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008956:Models, Chemical; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics; D010743:Phospholipids / Q000737:chemistry; D017434:Protein Structure, Tertiary / Q000502:physiology; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D013729:Terpenes / Q000737:chemistry; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D003432:Cross-Linking Reagents; D017261:Glycosylphosphatidylinositols; D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010743:Phospholipids; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D013729:Terpenes; D033921:Vesicular Transport Proteins; C017788:undecaprenol