Quantification of the Exchange of Subunits from Membrane Protein Complexes Using Foerster Transfer Recovery (FTR)

Abstract

To quantify the exchange of subunits of membrane protein complexes cells expressing CFP/YFP-tagged phospholamban (PLB) were observed by total internal reflection fluorescence (TIRF) microscopy. We performed YFP-selective photobleaching of spots, lines, or larger regions of interest on the basal surface of the cells. This resulted in enhanced CFP fluorescence, indicating CFP-YFP fluorescence resonance energy transfer (FRET). Subsequent spatial broadening of this “pseudo-photoactivated” CFP fluorescence was analyzed as a measure of the lateral diffusion of PLB complexes away from the target region. In addition, exchange of bleached and unbleached YFP-PLB from complexes restored FRET over time. This process of Foerster transfer recovery (FTR) was taken to indicate the rate of exchange of fluorescently-labeled subunits of the membrane protein complex. Diffusion and exchange processes were quantified by image analysis using a custom MatLab application for 2-dimensional Gaussian fitting. In addition to its application to FTR, this approach may be useful for cytoplasmic proteins as a way of quantifying dynamic membrane recruitment and lateral diffusion on the plane of the bilayer. Fig. 1 shows the diffusion of acceptor-photobleached CFP/YFP-PLB complexes from a target region, followed by subunit exchange.View Large Image | View Hi-Res Image | Download PowerPoint Slide