The length of the flexible SNAREpin juxtamembrane region is a critical determinant of SNARE-dependent fusion.
The topology of a SNARE complex bridging two docked vesicles could act as a mechanical couple to do work on the lipid bilayer resulting in fusion. To test this, we prepared a series of modified SNARE proteins and determined their effects on SNARE-dependent membrane fusion. When two helix-breaking proline residues are introduced into the juxtamembrane region of VAMP, there is little or no effect on fusion, and the same change in syntaxin 1A only reduced the extent and rate of fusion by half. The insertion of a flexible linker between the transmembrane domain and the conserved coiled-coil domain only moderately affected fusion; however, fusion efficiency systematically decreased with increasing length of the linker. Together, these results rule out a requirement for helical continuity and suggest that distance is a critical factor for membrane fusion.
McNew
J A
JA
Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Weber
T
T
Engelman
D M
DM
Söllner
T H
TH
Rothman
J E
JE
D000595:Amino Acid Sequence; D000954:Antigens, Surface / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D018583:Pliability; D011392:Proline / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D050683:R-SNARE Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D002352:Carrier Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D011994:Recombinant Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D011392:Proline