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Title: 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:
 [1];  [2];  [3];  [4];  [5];  [4];  [4];  [6];  [6];  [4]
  1. Department of Plant Biology, Carnegie Institution for Science, Stanford, United States, Institute of Plant Genetics, Italian National Research Council (CNR-IGV), Palermo, Italy
  2. Department of Plant Biology, Carnegie Institution for Science, Stanford, United States, NanoPolyPhotonik, Fraunhofer Institute for Applied Polymer Research, Potsdam-Golm, Germany
  3. Feedstocks Division, Joint BioEnergy Institute, Emeryville, United States
  4. Department of Plant Biology, Carnegie Institution for Science, Stanford, United States
  5. Department of Biochemistry, Institute for Organic Chemistry and Biochemistry, and BIOSS Center for Biological Signalling Studies, University of Freiburg, Freiburg, Germany
  6. 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:
Journal Article: 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. 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., and Frommer, Wolf B. Tue . "Fluorescent sensors reporting the activity of ammonium transceptors in live cells". United States. doi: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},
issn = {2050-084X},
number = ,
volume = 2,
place = {United States},
year = {2013},
month = {7}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.7554/eLife.00800

Citation Metrics:
Cited by: 20 works
Citation information provided by
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Works referenced in this record:

The Green Fluorescent Protein
journal, June 1998


Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators
journal, November 2009

  • Tian, Lin; Hires, S Andrew; Mao, Tianyi
  • Nature Methods, Vol. 6, Issue 12, p. 875-881
  • DOI: 10.1038/nmeth.1398

Circularly permuted green fluorescent proteins engineered to sense Ca2+
journal, March 2001

  • Nagai, T.; Sawano, A.; Park, E. S.
  • Proceedings of the National Academy of Sciences, Vol. 98, Issue 6, p. 3197-3202
  • DOI: 10.1073/pnas.051636098