GENETIC AND MORPHOLOGICAL ANALYSES REVEAL A CRITICAL INTERACTION BETWEEN THE C-TERMINI OF 2 SNARE PROTEINS AND A PARALLEL 4 HELICAL ARRANGEMENT FOR THE EXOCYTIC SNARE COMPLEX

In a screen fur suppressors of a temperature-sensitive mutation in the yeast SNAP-25 homolog, Sec9,,ve have identified a gain-of-function mu tation in the yeast synaptobrevin homolog, Snc2, The genetic propertie s of this suppression point to a specific interaction between the C-te rmini of Sec9 and Snc2 within the SNARE complex. Biochemical analysis of interactions between the wild-type and mutant proteins confirms thi s prediction, demonstrating specific effects of these mutations on int eractions between the SNAREs, The location of the mutations suggests t hat the C-terminal H2 helical domain of Sec9 is likely to be aligned i n parallel with Snc2 in the SNARE complex. To test this prediction, we examined the structure of the yeast exocytic SNARE complex by deep-et ch electron microscopy, Like the neuronal SNARE complex, it is a rod s imilar to 14 nm long. Using epitope tags, antibodies and maltose-bindi ng protein markers, we find that the helical domains of Sso, Snc and b oth halves of Sec9 are all aligned, in parallel within the SNARE compl ex, suggesting that tbe yeast exocytic SNARE complex consists of a par allel four helix bundle. Finally, we find a similar arrangement for SN AP-25 in the neuronal SNARE complex. This provides strong evidence tha t the exocytic SNARE complex is a highly conserved structure composed of four parallel helical domains whose C-termini must converge in orde r to bring about membrane fusion.