Intracellular interaction between syntaxin and Munc 18-1 revealed by fluorescence resonance energy transfer.
Neurosecretion is catalyzed by assembly of a soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-complex composed of SNAP-25, synaptobrevin and syntaxin. Munc 18-1 is known to bind to syntaxin in vitro. This interaction prevents assembly of the SNARE-complex, but might also affect intracellular targeting of the proteins. We have fused syntaxin and Munc 18 to the yellow- (YFP) or cyan-fluorescence-protein (CFP) and expressed the constructs in CHO- and MDCK-cells. We have studied their localization with confocal microscopy and a possible protein-protein interaction with fluorescence-resonance energy transfer (FRET). YFP-syntaxin localizes to intracellular membranes. CFP-Munc 18 is present in the cytoplasm as expected for a protein lacking membrane targeting domains. However, Munc 18 is redirected to internal membranes when syntaxin is coexpressed, but only limited transport of the proteins to the plasma membrane was observed. An interaction between Munc 18 and syntaxin could be demonstrated by FRET using two methods, sensitized acceptor fluorescence and acceptor photobleaching. A mutation in syntaxin (L165A, E166A), which is known to inhibit binding to Munc 18 in vitro, prevents colocalization of the proteins and also the FRET signal. Thus, a protein-protein interaction between Munc 18 and syntaxin occurs on intracellular membranes, which is required but not sufficient for quantitative transport of both proteins to the plasma membrane.
Yerrapureddy
Adinarayanareddy
A
Department of Immunology and Molecular Biology, Veterinary Faculty, Free University, Berlin. mveit@zedat.fu-berlin.de
Korte
Thomas
T
Hollmann
Susanne
S
Nordhoff
Marcel
M
Ahnert-Hilger
Gudrun
G
Herrmann
Andreas
A
Veit
Michael
M
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D018613:Microscopy, Confocal; D051938:Munc18 Proteins / Q000032:analysis / Q000378:metabolism*; D016716:PC12 Cells; D025941:Protein Interaction Mapping; D021122:Protein Subunits / Q000378:metabolism; D050765:Qa-SNARE Proteins / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D014162:Transfection
D051938:Munc18 Proteins; D021122:Protein Subunits; D050765:Qa-SNARE Proteins; C497939:Stxbp1 protein, rat; D049452:Green Fluorescent Proteins