Definition of a minimal munc18c domain that interacts with syntaxin 4.
munc18c is a critical protein involved in trafficking events associated with syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its interaction with syntaxin 4, fragments of munc18c were generated and their interaction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. In 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 munc18c(226-592), munc18c(1-100), munc18c(43-139) or munc18c(66-139), interacted with the cytoplasmic portion 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 syntaxin 4, Stx4(29-157), failed to interact with full-length munc18c(1-592), indicating that a larger portion of syntaxin 4 is necessary for the interaction. The yeast two-hybrid results were confirmed by protein-protein interaction studies between Stx4(2-273) and glutathione S-transferase fusion proteins 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 with 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 syntaxin 4.
Grusovin
J
J
CSIRO Health Sciences and Nutrition, 343 Royal Parade, Parkville 3052, Victoria, Australia.
Stoichevska
V
V
Gough
K H
KH
Nunan
K
K
Ward
C W
CW
Macaulay
S L
SL
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D050765:Qa-SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins; D001616:beta-Galactosidase / Q000378:metabolism
D017931:DNA Primers; D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D050765:Qa-SNARE Proteins; C497943:Stxbp3 protein, mouse; D033921:Vesicular Transport Proteins; D001616:beta-Galactosidase