Fluorescent sensors reporting the activity of ammonium transceptors in live cells
Abstract
Ammonium serves as key nitrogen source and metabolic intermediate, yet excess causes toxicity. Ammonium uptake is mediated by ammonium transporters, whose regulation is poorly understood. While transport can easily be characterized in heterologous systems, measuring transporter activity in vivo remains challenging. Here we developed a simple assay for monitoring activity in vivo by inserting circularly-permutated GFP into conformation-sensitive positions of two plant and one yeast ammonium transceptors (‘AmTrac’ and ‘MepTrac’). Addition of ammonium to yeast cells expressing the sensors triggered concentration-dependent fluorescence intensity (FI) changes that strictly correlated with the activity of the transporter. Fluorescence-based activity sensors present a novel technology for monitoring the interaction of the transporters with their substrates, the activity of transporters and their regulation in vivo, which is particularly valuable in the context of analytes for which no radiotracers exist, as well as for cell-specific and subcellular transport processes that are otherwise difficult to track.
- Authors:
-
- Department of Plant Biology, Carnegie Institution for Science, Stanford, United States, Institute of Plant Genetics, Italian National Research Council (CNR-IGV), Palermo, Italy
- Department of Plant Biology, Carnegie Institution for Science, Stanford, United States, NanoPolyPhotonik, Fraunhofer Institute for Applied Polymer Research, Potsdam-Golm, Germany
- Feedstocks Division, Joint BioEnergy Institute, Emeryville, United States
- Department of Plant Biology, Carnegie Institution for Science, Stanford, United States
- Department of Biochemistry, Institute for Organic Chemistry and Biochemistry, and BIOSS Center for Biological Signalling Studies, University of Freiburg, Freiburg, Germany
- Department of Physical Chemistry, Institute of Chemistry, University of Potsdam, Potsdam, Germany
- Publication Date:
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 1197684
- Alternate Identifier(s):
- OSTI ID: 1197685
- Grant/Contract Number:
- AC02-05CH11231
- Resource Type:
- Published Article
- Journal Name:
- eLife
- Additional Journal Information:
- Journal Name: eLife Journal Volume: 2; Journal ID: ISSN 2050-084X
- Publisher:
- eLife Sciences Publications, Ltd.
- Country of Publication:
- United States
- Language:
- English
Citation Formats
De Michele, Roberto, Ast, Cindy, Loqué, Dominique, Ho, Cheng-Hsun, Andrade, Susana LA, Lanquar, Viviane, Grossmann, Guido, Gehne, Sören, Kumke, Michael U., and Frommer, Wolf B. Fluorescent sensors reporting the activity of ammonium transceptors in live cells. United States: N. p., 2013.
Web. doi:10.7554/eLife.00800.
De Michele, Roberto, Ast, Cindy, Loqué, Dominique, Ho, Cheng-Hsun, Andrade, Susana LA, Lanquar, Viviane, Grossmann, Guido, Gehne, Sören, Kumke, Michael U., & Frommer, Wolf B. Fluorescent sensors reporting the activity of ammonium transceptors in live cells. United States. https://doi.org/10.7554/eLife.00800
De Michele, Roberto, Ast, Cindy, Loqué, Dominique, Ho, Cheng-Hsun, Andrade, Susana LA, Lanquar, Viviane, Grossmann, Guido, Gehne, Sören, Kumke, Michael U., and Frommer, Wolf B. Tue .
"Fluorescent sensors reporting the activity of ammonium transceptors in live cells". United States. https://doi.org/10.7554/eLife.00800.
@article{osti_1197684,
title = {Fluorescent sensors reporting the activity of ammonium transceptors in live cells},
author = {De Michele, Roberto and Ast, Cindy and Loqué, Dominique and Ho, Cheng-Hsun and Andrade, Susana LA and Lanquar, Viviane and Grossmann, Guido and Gehne, Sören and Kumke, Michael U. and Frommer, Wolf B.},
abstractNote = {Ammonium serves as key nitrogen source and metabolic intermediate, yet excess causes toxicity. Ammonium uptake is mediated by ammonium transporters, whose regulation is poorly understood. While transport can easily be characterized in heterologous systems, measuring transporter activity in vivo remains challenging. Here we developed a simple assay for monitoring activity in vivo by inserting circularly-permutated GFP into conformation-sensitive positions of two plant and one yeast ammonium transceptors (‘AmTrac’ and ‘MepTrac’). Addition of ammonium to yeast cells expressing the sensors triggered concentration-dependent fluorescence intensity (FI) changes that strictly correlated with the activity of the transporter. Fluorescence-based activity sensors present a novel technology for monitoring the interaction of the transporters with their substrates, the activity of transporters and their regulation in vivo, which is particularly valuable in the context of analytes for which no radiotracers exist, as well as for cell-specific and subcellular transport processes that are otherwise difficult to track.},
doi = {10.7554/eLife.00800},
journal = {eLife},
number = ,
volume = 2,
place = {United States},
year = {Tue Jul 02 00:00:00 EDT 2013},
month = {Tue Jul 02 00:00:00 EDT 2013}
}
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https://doi.org/10.7554/eLife.00800
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