Quantification of Extracellular Ammonium Concentrations and Transporter Activity in Yeast Using AmTrac Fluorescent Sensors

Abstract

AmTracs are the first example of “activity sensors”, since they report the activity of ammonium transporters by means of fluorescence readout in vivo (De Michele et al., 2013). AmTracs are based on a single fluorescent protein, a circularly permuted GFP (cpGFP), inserted into the cytosolic loop connecting the two pseudosymmetrical halves of the Arabidopsis and yeast plasma membrane ammonium transporters AtAMTs and ScMEP (Figure 1). Recently, FRET-based activity sensors for nitrate and peptide transporters have also been developed (Ho et al., 2014). Since transporter activity directly depends on the availability of substrate, AmTracs measure extracellular ammonium concentrations. Several versions of AmTrac exist, with different fluorescence intensity (FI) responses and affinities for ammonium, and based on different ammonium transporters (AmTrac: AtAMT1;3; AmTrac1;2: At AMT1;2; MepTrac: ScMEP2). Currently, the most useful AmTrac versions are probably AmTrac-GS (bright, with Km of 50 µM) and AmTrac-100 (a high capacity version with Km of 100 µM). The protocol for measuring ammonium concentrations in yeast cells at the fluorimeter is the same for all versions.Figure 1. Model of AmTrac/MepTrac sensors and AMT transport mechanism. We propose that AMT switches between at least two distinct states during transport of ammonium: An outward, open state A and an inward, open state B. The movement of TMH-V (red) and TMH-VI (blue) is transmitted to the connecting loop, affecting the inserted cpGFP (green) and resulting in a change in fluorescence emission.