Design and Functional Analysis of Fluorescent Nitrate and Peptide Transporter Activity Sensors in Yeast Cultures

Abstract

This protocol describes the methods used to engineer and deploy genetically encoded fluorescence activity reporters for nitrate and peptide transporter activity in yeast cells. Fusion of the dual-affinity nitrate transceptor CHL1/AtNRT1.1/AtNPF6.3 or four different peptide transporters (AtPTR1, 2, 4, and 5) from Arabidopsis to a pair of fluorescent proteins with different spectral properties, enabled us to engineer the NiTracs (nitrate transporter activity tracking sensors) and the PepTracs (peptide transporter activity tracking sensors), ratiometric fluorescence activity sensors that monitor the activity of the plasma membrane nitrate transceptor or the peptide transporters in vivo (Ho et al., 2014). The NiTrac1 sensor responds specifically and reversibly to the addition of nitrate, while the PepTracs respond to addition of dipeptides, either by a reduction in donor and acceptor emission, while acceptor-excited emission remains unaltered, or by a change in ratio of the fluorophore emission. All sensors are suitable for ratiometric imaging. The similarity of the biphasic kinetics of the NiTrac1 sensor response [from µM to mM (Liu and Tsay, 2003)] and the nitrate transport kinetics of the native nitrate transceptor, intimates that NiTrac1 provides information on conformational rearrangements during the transport cycle, thereby reporting transporter activity over a wide range of external nitrate concentrations. Several variants of NiTrac have been engineered, which differ with respect to their affinity for nitrate (NiTrac1: CHL1; NiTracT101A: CHL1T101A). NiTrac also recognizes chlorate. Here we describe a simple method for the design, implementation, and detection of nitrate transceptor activity in yeast cells using a spectrofluorimeter.