Analysis of the Munc18b-syntaxin binding interface. Use of a mutant Munc18b to dissect the functions of syntaxins 2 and 3.
Munc18b is a mammalian Sec1-related protein that is abundant in epithelial cells and regulates vesicle transport to the apical plasma membrane. We constructed a homology model of Munc18b in complex with syntaxin 3 based on the crystal structure of the neuronal Sec1.syntaxin 1A complex. In this model we identified all residues in the interface between the two proteins that contribute directly to the interaction and mutagenized residues in Munc18b to alter its binding to syntaxins 1A, 2, and 3. The syntaxin-binding properties of the mutants were tested using an in vitro assay and by a co-immunoprecipitation approach employing Munc18b expressed in CHO-K1 cells. Three Munc18b variants, W28S, S42K, and E59K, were generated that are defective in binding to all three syntaxins. A fourth mutant protein, S48D, shows abolishment of syntaxin 3 interaction but binds syntaxin 2 at normal and syntaxin 1A at mildly reduced efficiency. Over-expression of Munc18b S48D inhibited transport of influenza hemagglutinin to the apical surface of Madin-Darby canine kidney II cells, which express syntaxin 2 abundantly, but not of Caco-2 cells, in which syntaxin 3 is the major apical target SNARE (soluble NSF (N-ethylmaleimide sensitive factor) attachment protein receptors). This suggests that, although syntaxin 3 is the main target SNARE operating in exocytic transport to the apical plasma membrane in certain epithelial cell types, syntaxin 2 may play an important role in this trafficking route in others.
Kauppi
Maria
M
Department of Molecular Medicine, National Public Health Institute, Biomedicum, P.O. Box 104, FIN-00251 Helsinki, Finland.
Wohlfahrt
Gerd
G
Olkkonen
Vesa M
VM
D000595:Amino Acid Sequence; D000818:Animals; D000954:Antigens, Surface / Q000502:physiology*; D001692:Biological Transport; D016466:CHO Cells; D018938:Caco-2 Cells; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D014644:Genetic Variation; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D051938:Munc18 Proteins; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D050827:Syntaxin 1; D014162:Transfection; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D018076:DNA, Complementary; D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; C494212:STX1A protein, human; C497940:STXBP2 protein, human; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins