Fluorescent sensors for activity and regulation of the nitrate transceptor CHL1/NRT1.1 and oligopeptide transporters

Abstract

To monitor nitrate and peptide transport activity in vivo, we converted the dual-affinity nitrate transceptor CHL1/NRT1.1/NPF6.3 and four related oligopeptide transporters PTR1, 2, 4, and 5 into fluorescence activity sensors (NiTrac1, PepTrac). Substrate addition to yeast expressing transporter fusions with yellow fluorescent protein and mCerulean triggered substrate-dependent donor quenching or resonance energy transfer. Fluorescence changes were nitrate/peptide-specific, respectively. Like CHL1, NiTrac1 had biphasic kinetics. Mutation of T101A eliminated high-affinity transport and blocked the fluorescence response to low nitrate. NiTrac was used for characterizing side chains considered important for substrate interaction, proton coupling, and regulation. We observed a striking correlation between transport activity and sensor output. Coexpression of NiTrac with known calcineurin-like proteins (CBL1, 9; CIPK23) and candidates identified in an interactome screen (CBL1, KT2, WNKinase 8) blocked NiTrac1 responses, demonstrating the suitability for in vivo analysis of activity and regulation. The new technology is applicable in plant and medical research.DOI:http://dx.doi.org/10.7554/eLife.01917.001

Metabolic engineering to enhance the value of plants as green factories

“…While cellular/subcellular fractionation followed by metabolite analysis continues to be a valuable tool to determine where metabolites accumulate, a new generation of approaches is rapidly developing. For example, fluorescence resonance energy transfer (FRET)-based nanosensors have been successfully used to measure subcellular levels of a number of metabolites and other small molecules, including maltose, glucose, sucrose, ammonium and nitrate in plants and other organisms (De Michele et al, 2013;Fehr et al, 2002;Fehr et al, 2004;Fehr et al, 2005;Frommer et al, 2009;Ho and Frommer, 2014;Jones et al, 2013). The sensitivity of nanosensors is in the nano-to micromolar range, and they provide significant advantages over other quantitative reporters of small molecule concentration inside cells (Okumoto et al, 2012).…”

Modulation of ABA responses by the protein kinase WNK8

“…Based on the analyses of loss‐of‐function mutants, WNKs appeared to be involved in the regulation of flowering time, glucose, drought, and abscisic acid (ABA) responses . WNK8 is the best studied family member that binds to and phosphorylates the vacuolar H + ‐ATPase subunit VHA‐C , the nitrate transceptor NRT1.1 , the transcriptional regulator EDM2 , the regulator of G‐protein signaling RGS1 , and the scaffold protein RACK1 . Among these, RACK1 proteins have been implicated as negative regulators of ABA responses .…”

Seeing is better than believing: visualization of membrane transport in plants

“…A better proxy might be the recently developed transport activity sensors, such as AmTrac and MepTrac [13,14], NiTrac1 and PepTrac [15]. Although these transport activity sensors do not strictly monitor transport, and have not yet been tested in planta, they will most likely become valuable, especially in the context of analytes for which no tracers exist.…”