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 t heir effects on SNARE-dependent membrane fusion. When two helix-breaking pr oline 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 r educed the extent and rate of fusion by half. The insertion of a flexible l inker 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 ru le out a requirement for helical continuity and suggest that distance is a critical factor for membrane fusion.