Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p.
The fusion of intracellular transport vesicles with their target membranes requires the assembly of SNARE proteins anchored in the apposed membranes. Here we use recombinant cytoplasmic domains of the yeast SNAREs involved in Golgi to plasma membrane trafficking to examine this assembly process in vitro. Binary complexes form between the target membrane 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-helical structure, indicating that folding and complex formation are linked. Surprisingly, we find that binary complex formation is extremely slow, with a second-order rate constant of approximately 3 M(-1) s(-1). An N-terminal regulatory domain of Sso1p accounts for slow assembly, since in its absence complexes assemble 2,000-fold more rapidly. Once binary complexes form, ternary complex formation is rapid and is not affected by the presence of the regulatory domain. Our results imply that proteins that accelerate SNARE assembly in vivo act by relieving inhibition by this regulatory domain.
Nicholson
K L
KL
Department of Chemistry, Princeton University, New Jersey 08544, USA.
Munson
M
M
Miller
R B
RB
Filip
T J
TJ
Fairman
R
R
Hughson
F M
FM
D002942:Circular Dichroism; D004355:Drug Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008561:Membrane Fusion / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins
D005656:Fungal Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010446:Peptide Fragments; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; C084006:SSO1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins