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 (Mint3 Delta 6) is able to interact with the constitutively active form of Rab6A. Mint3 Delta 4, 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 FAGS-based FRET analysis is a suitable method for interaction studies between,two proteins in living cells. (C) 2010 Elsevier Inc. All rights reserved.