FRET-FLIM to determine protein interactions and membrane topology of enzyme complexes

Determining protein-protein interactions is vital for gaining knowledge on cellular and metabolic processes including enzyme complexes and metabolons. Förster resonance energy transfer together with fluorescence lifetime imaging microscopy (FRET-FLIM) is an advanced imaging methodology that allows for the quantitative detection of protein-protein interactions. In this method, proteins of interest for interaction studies are fused to different fluorophores such as eGFP (donor molecule) and mRFP (acceptor molecule). Energy transfer between the two fluorophore groups can only occur efficiently when the proteins of interest are in close physical proximity around 10 nm or less and therefore are most likely interacting. FRET-FLIM measures the decrease in excited state lifetime of the donor fluorophore (eGFP) with and without the presence of the acceptor (mRFP), and can therefore give information on protein-protein interactions as well as the membrane topology of the tested protein. Here we describe the production of fluorescent protein fusions for FRET-FLIM analysis in tobacco leaf epidermal cells using Agrobacterium-mediated plant transformation as well as a FRET-FLIM data acquisition and analysis protocol in plant cells. These protocols are applicable and can be adapted for both membrane and soluble proteins in different cellular localizations