1. Structural Biology and Molecular Biophysics
  2. Cell Biology

Single-fluorophore membrane transport activity sensors with dual-emission read-out

  1. Cindy Ast
  2. Roberto De Michele
  3. Michael U Kumke
  4. Wolf B Frommer  Is a corresponding author
  1. Carnegie Institution for Science, United States
  2. Italian National Research Council, Italy
  3. University of Potsdam, Germany
https://doi.org/10.7554/eLife.07113
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  1. Cindy Ast
  2. Roberto De Michele
  3. Michael U Kumke
  4. Wolf B Frommer
(2015)
Single-fluorophore membrane transport activity sensors with dual-emission read-out
eLife 4:e07113.
https://doi.org/10.7554/eLife.07113
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Abstract

We recently described a series of genetically encoded, single-fluorophore-based sensors, termed AmTrac and MepTrac, which monitor membrane transporter activity in vivo (De Michele et al., 2013). However, being intensiometric, AmTrac and Meptrac are limited in their use for quantitative studies. Here, we characterized the photophysical properties (steady-state and time-resolved fluorescence spectroscopy as well as anisotropy decay analysis) of different AmTrac sensors with diverging fluorescence properties in order to generate improved, ratiometric sensors. By replacing key amino acid residues in AmTrac we constructed a set of dual-emission AmTrac sensors named deAmTracs. deAmTracs show opposing changes of blue and green emission with almost doubled emission ratio upon ammonium addition. The response ratio of the deAmTracs correlated with transport activity in mutants with altered capacity. Our results suggest that partial disruption of distance-dependent excited-state proton transfer (ESPT) is important for the successful generation of single-fluorophore-based dual-emission sensors.

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Author details

  1. Cindy Ast

    Department of Plant Biology, Carnegie Institution for Science, Stanford California, United States
    Competing interests
    The authors declare that no competing interests exist.

  2. Roberto De Michele

    Institute of Biosciences and Bioresources, Italian National Research Council, Palermo, Italy
    Competing interests
    The authors declare that no competing interests exist.

  3. Michael U Kumke

    Department of Physical Chemistry, Institute of Chemistry, University of Potsdam, Potsdam, Germany
    Competing interests
    The authors declare that no competing interests exist.

  4. Wolf B Frommer

    Department of Plant Biology, Carnegie Institution for Science, Stanford California, United States
    For correspondence
    wfrommer@carnegiescience.edu
    Competing interests
    The authors declare that no competing interests exist.

Copyright

© 2015, Ast et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Cindy Ast
  2. Roberto De Michele
  3. Michael U Kumke
  4. Wolf B Frommer
(2015)
Single-fluorophore membrane transport activity sensors with dual-emission read-out
eLife 4:e07113.
https://doi.org/10.7554/eLife.07113

Share this article

https://doi.org/10.7554/eLife.07113

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Further reading

    1. Cell Biology
    2. Plant Biology

    Fluorescent sensors reporting the activity of ammonium transceptors in live cells

    Roberto De Michele, Cindy Ast ... Wolf B Frommer
    Research Article

    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.