Interaction of Munc-18-2 with syntaxin 3 controls the association of apical SNAREs in epithelial cells.
The docking/fusion of transport vesicles mediated by the soluble NSF attachment protein receptors (SNAREs) is thought to be regulated by Sec1-related proteins. Munc-18-2, a member of this family, is predominantly expressed in the epithelial cells of several tissues. We demonstrate here that Munc-18-2 colocalizes with syntaxin 3 at the apical plasma membrane of intestinal epithelium and Caco-2 cells. The presence of a physical complex of the two proteins is verified by 2-way coimmunoprecipitation. The quantity of the complex is reduced by treatment of Caco-2 cells with the alkylating agent N-ethylmaleimide which also has an inhibitory effect on the ability of Munc-18-2 to associate with syntaxin 3 in vitro. The amount of Munc-18-2 in the complex increases upon treatment of the cells with the protein kinase C activator phorbol myristate acetate, indicating a functional connection between the complex and cell signalling. Increasing the amount of Munc-18-2 bound to syntaxin 3 by overexpression results in a marked decrease in the SNARE proteins SNAP-23 and cellubrevin bound to the syntaxin. These results define a novel functional complex of Munc-18-2 and syntaxin 3 involved in the regulation of apical membrane transport.
Riento
K
K
Department of Biochemistry, National Public Health Institute, Mannerheimintie 166, FIN-00300, Helsinki, Finland.
Galli
T
T
Jansson
S
S
Ehnholm
C
C
Lehtonen
E
E
Olkkonen
V M
VM
D000818:Animals; D001692:Biological Transport; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D004285:Dogs; D015870:Gene Expression; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D011506:Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050600:SNARE Proteins; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection; D051040:Vesicle-Associated Membrane Protein 3; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; C100493:SNAP23 protein, human; D050600:SNARE Proteins; C497940:STXBP2 protein, human; D051040:Vesicle-Associated Membrane Protein 3; D033921:Vesicular Transport Proteins