Characterizing the interaction between the Rab6 GTPase and Mint3 via flow cytometry based FRET analysis.
In extension to previously applied techniques like yeast two-hybrid and GST pull-down assays, we successfully established a FACS-based FRET analysis to investigate the interaction of the Mint3 adaptor protein and the small Rab GTPase Rab6A in living mammalian cells. A Mint3 mutant containing only the PTB domain (Mint3Delta6) is able to interact with the constitutively active form of Rab6A. Mint3Delta4, a mutant lacking part of the PTB domain was unable to interact with Rab6A in GST pull-down analysis and did not produce FRET signals, when co-expressed with active Rab6A. We demonstrate that this FACS-based FRET analysis is a suitable method for interaction studies between two proteins in living cells.
Thyrock
Anika
A
Department of Experimental Tumorbiology, University Muenster, Badestr 9, D-48149 Muenster, Germany. a.thyrock@uni-muenster.de
Stehling
Martin
M
Waschbüsch
Dieter
D
Barnekow
Angelika
A
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002469:Cell Separation; D005434:Flow Cytometry / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D025941:Protein Interaction Mapping / Q000379:methods*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
C117536:APBA3 protein, human; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C094135:Rab6 protein; D020691:rab GTP-Binding Proteins