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

BioRxiv : the Preprint Server for Biology
Cheng-Hsun Ho, W B Frommer


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.

Related Concepts

NRT1.1 protein, Arabidopsis
Fluorescence Resonance Energy Transfer
Protein Transport
ATP Synthesis Coupled Proton Transport
Protein Expression

Related Feeds

BioRxiv & MedRxiv Preprints

BioRxiv and MedRxiv are the preprint servers for biology and health sciences respectively, operated by Cold Spring Harbor Laboratory. Here are the latest preprint articles (which are not peer-reviewed) from BioRxiv and MedRxiv.