Publikationsdatenbank

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

Bandzählung: 22

Heftzählung: 5

Seiten: 401 – 410

ISSN: 0968-7688

Sprache

Englisch

Verweise

Pubmed: 16308274

Kontakt

Institut für Mikrobiologie und Tierseuchen

Robert-von-Ostertag-Str. 7-13
Gebäude 35
14163 Berlin
+49 30 838 51840 / 51843
mikrobiologie@vetmed.fu-berlin.de

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.