Determining protein-protein interactions is vital for gaining knowledge on cellular and metabolic processes including enzyme complexes and metabolons. Förster resonance energy transfer 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 enhanced green fluorescent protein (eGFP; donor molecule) and monomeric red fluorescent protein (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, 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 and 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 and 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. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Protein expression in tobacco leaf cells via transient Agrobacterium-mediated plant transformation Basic Protocol 2: FRET-FLIM data acquisition and analysis.
Keywords: FRET-FLIM; eGFP; endoplasmic reticulum; mRFP; membrane protein; protein-protein interactions; topology.
© 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.