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    Intracellular interaction between syntaxin and Munc 18-1 revealed by fluorescence resonance energy transfer (2005)

    Art
    Zeitschriftenartikel / wissenschaftlicher Beitrag
    Autoren
    Yerrapureddy, Adinarayanareddy
    Korte, Thomas
    Hollmann, Susanne
    Nordhoff, Marcel
    Ahnert-Hilger, Gudrun
    Herrmann, Andreas
    Veit, Michael
    Quelle
    Molecular membrane biology : MMB; 22(5) — S. 401–410
    ISSN: 0968-7688
    Sprache
    Englisch
    Verweise
    Pubmed: 16308274
    Kontakt
    Institut für Immunologie

    Robert-von-Ostertag-Str. 7-13
    Gebäude 35
    14163 Berlin
    +49 30 838 51834

    Abstract / Zusammenfassung

    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.