Fluorescent sensors reporting the activity of ammonium transceptors in live cells
- Carnegie Institution for Science, United States
- Institute of Plant Genetics, Italian National Research Council (CNR-IGV), Italy
- Fraunhofer Institute for Applied Polymer Research, Germany
- Joint BioEnergy Institute, United States
- Institute for Organic Chemistry and Biochemistry, and BIOSS Center for Biological Signalling Studies, University of Freiburg, Germany
- Institute of Chemistry, University of Potsdam, Germany
Figures
Figure 1
Design of fusion constructs.
(A) Topological representation of AMT1;3 by HMMTOP (Tusnady and Simon, 2001). Eleven TMH are organized in a pseudo-symmetric structure (TMH I-V and TMH VI-X) with an extra terminal TMH-XI that …
Figure 2
Development of AmTrac.
(A) Growth of the yeast Δmep1,2,3 mutant transformed with fusion variants on solid media containing 2 mM NH4Cl or 1 mM arginine (growth control) as the sole nitrogen source for 3 days. Composition …
Figure 3
Effect of L5 on transport and fluorescence response.
(A) Amino acid sequence of the L5–6 region of AMT1;3. Residues in ‘blue’ correspond to TMH 5 (left) and TMH 6 (right). (B) Influence of the insertion position of mcpGFP into L5 on transport activity …
Figure 4
Characterization of AmTrac transport and responses.
(A) Currents recorded in single oocytes injected with water, AMT1;3, AmTrac, or AmTrac-LS, and perfused with NH4Cl at the indicated concentrations. (B) Kinetics of NH4+-induced currents of AMT1;3, …
Figure 5
Development of improved variants of AmTrac.
(A) Screen for improved sensor variants. Fluorescence intensity and fluorescence response of ∼350 random variants of the C-terminal linker of mcpGFP to addition of 1 mM NH4Cl. The brightest and most …
Figure 6
AmTrac mutant analysis.
(A) Confocal z-section of yeast expressing AmTrac or its inactive variants carrying mutations D202N, G460D or T464D. Bar 10 µm. (B) Growth complementation of the Δmep1,2,3 or wt yeast expressing …
Figure 7
Suppressor mutants restore transport and fluorescence response.
(A) Growth of the yeast Δmep1,2,3 strain transformed with suppressor mutants and grown on solid media containing the indicated concentrations of NH4Cl, (NH4)2SO4 (as anion control) or 1 mM arginine …
Figure 8
Characterization of a high capacity sensor variant.
(A) Kinetics of NH4+-induced currents of AmTrac-100. The Km was 3.4 ± 0.56 mM. The data were fitted to Michaelis–Menten kinetics. Oocytes were clamped at −120 mV (independent data from three …
Figure 9
AmTrac1;2 and MepTrac response.
(A) Growth complementation of Δmep1,2,3 yeast expressing MEP2, MepTrac, their H194E mutants or AmTrac1;2 on solid media containing 2 mM NH4Cl or 1 mM arginine (growth control) as sole nitrogen …
Tables
Table 1
List of suppressor mutations, their location and frequency
| Mutation | Location | # | % |
|---|---|---|---|
| Q61E | TM 1 | 3 | 5 |
| Q61H | TM 1 | 5 | 9 |
| F64S | TM 1 | 3 | 5 |
| I140S | TM 3 | 1 | 2 |
| A141E | TM 3 | 26 | 46 |
| A142P | TM 3 | 13 | 23 |
| I150T | TM 3 | 2 | 4 |
| D464V | C-tail | 3 | 5 |
| Total | 56 | 100 |
Additional files
-
Supplementary file 1
Primers used for AmTrac and MepTrac cloning.
- https://doi.org/10.7554/eLife.00800.013
