Characterization of improved L‐tryptophan FRET biosensor for measurement of real time transport in live cells

Abstract

The transmembrane flux and exchange of amino acids is mediated by various cooperating transporters that control cell and body amino acid homeostasis. To characterize the function of specific amino acid transporters and their cooperation in live cells, we aim at measuring subcellular changes in amino acid concentration in real time. To generate a highly sensitive biosensor, we modified a previously described L-tryptophan FRET biosensor composed of CFP and YFP linked together by a bacterial L-tryptophan binding protein and expressed this new CTY in wild type COS-7 cells. These cells display high endogenous levels of the amino acid antiporter LAT1-4F2 (SLC7A5-SLC3A2), a high affinity exchanger of L-tryptophan and of other neutral amino acids. Additionally, we expressed the low affinity aromatic amino acid uniporter TAT1 (SLC16A10) that can cooperate with Lat1-4F2hc. As a low affinity facilitated diffusion pathway, TAT1 can also be used for the control of intracellular L-tryptophan concentration and sensor calibration. Using a microplate reader and live cell imaging FRET, we show that the new biosensor displays a much improved FRET efficiency compared with the previously described CTYT. Thus, the new CTY biosensor allows us to understand better how the cooperation of amino acid exchangers (antiporters) and facilitated diffusion pathways (uniporters) can together control cellular amino acid homeostasis. Supported by Swiss NSF grant 31-130471 to FV