REGULATION OF SNARE COMPLEX ASSEMBLY BY AN N-TERMINAL DOMAIN OF THE T-SNARE SSO1P

The fusion of intracellular transport vesicles with their target membr anes requires the assembly of SNARE proteins anchored in the apposed m embranes. Here we use recombinant cytoplasmic domains of the yeast SNA REs involved in Golgi to plasma membrane trafficking to examine this a ssembly process in vitro. Binary complexes form between the target mem brane SNAREs Sso1p and Sec9p; these binary complexes can subsequently bind to the vesicle SNARE Snc2p to form ternary complexes. Binary and ternary complex assembly are accompanied by large increases in alpha-h elical structure, indicating that folding and complex formation are li nked. Surprisingly, we find that binary complex formation is extremely slow, with a second-order rate constant of similar to 3 M-1 s(-1). An N-terminal regulatory domain of Sso1p accounts for slow assembly, sin ce in its absence complexes assemble 2,000-fold more rapidly. Once bin ary complexes form, ternary complex formation is rapid and is not affe cted by the presence of the regulatory domain. Our results imply that proteins that accelerate SNARE assembly in vivo act by relieving inhib ition by this regulatory domain.