Topological restriction of SNARE-dependent membrane fusion

To fuse transport vesicles with target membranes, proteins of the SNARE (so luble N-ethylmaleimide-sensitive factor attachment protein receptors) compl ex must be located on both the vesicle (v-SNARE) and the target membrane (t -SNARE)(1). In yeast, four integral membrane proteins, Sed5, Bos1, Sec22 an d Bet1 (refs 2-6), each probably contribute a single helix to form the SNAR E complex that is needed for transport from endoplasmic reticulum to Golgi( 7-11). This generates a four-helix bundle(12), which ultimately mediates th e actual fusion event(13). Here we explore how the anchoring arrangement of the four helices affects their ability to mediate fusion. We reconstituted two populations of phospholipid bilayer vesicles, with the individual SNAR E proteins distributed in all possible combinations between them. Of the ei ght non-redundant permutations of four subunits distributed over two vesicl e populations, only one results in membrane fusion. Fusion only occurs when the v-SNARE Bet1 is on one membrane and the syntaxin heavy chain Sed5 and its two light chains, Bos1 and Sec22, are on the other membrane where they form a functional t-SNARE. Thus, each SNARE protein is topologically restri cted by design to function either as a v-SNARE or as part of a t-SNARE comp lex.