Definition of a minimal munc18c domain that interacts with syntaxin 4

munc18c is a critical protein involved in trafficking events associated wit h syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its in teraction with syntaxin 4, fragments of munc18c were generated and their in teraction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. I n vitro protein-protein interaction studies were then used to confirm that the interaction between the proteins was direct. Full-length munc18c(1-592) , munc18c(1-139) and munc18c(1-225), but not munc1.8c(226-592), munc18c(1-1 00), munc18c(43-139) or munc18c(66-139), interacted with the cytoplasmic po rtion of syntaxin 4, StX4(2-273), as assessed by yeast two-hybrid assay of growth on nutritionally deficient media and by beta-galactosidase reporter induction. The N-terminal predicted helix-a-helix-b-helix-c region of synta xin 4, Stx4(29-157), failed to interact with full-length munc18c(1-592), in dicating that a larger portion of syntaxin 4 is necessary for the interacti on. The yeast two-hybrid results were confirmed by protein-protein interact ion studies between Stx4(2-273) and glutathione S-transferase fusion protei ns of munc18c. Full-length munc18c(1-592), munc18c(1-139) and munc18c(1-225 ) interacted with Stx4(2-273) whereas munc18c(1-100) did not, consistent wi th the yeast two-hybrid data. These data thus identify a region of munc18c between residues 1 and 139 as a minimal domain for its interaction with syn taxin 4.