title: 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.
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, United States.
authors.forename: Cheng-Hsun
authors.initials: CH
authors.lastname: Ho
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, United States.
authors.forename: Wolf B
authors.initials: WB
authors.lastname: Frommer
mesh_terms: D000818:Animals; D027321:Anion Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D015374:Biosensing Techniques; D005260:Female; D005470:Fluorometry; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009566:Nitrates / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D009865:Oocytes; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014982:Xenopus laevis; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
chemical_list: D027321:Anion Transport Proteins; D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; C101492:NRT1.1 protein, Arabidopsis; D009566:Nitrates; D009842:Oligopeptides; D010940:Plant Proteins; C495664:Ptr1 protein, Arabidopsis; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria
keywords: FRET; biosensor; fluorescence; nitrate; oligopeptide; transport