Fluorescent sensors for activity and regulation of the nitrate transceptor CHL1/NRT1.1 and oligopeptide transporters

  1. Cheng-Hsun Ho
  2. Wolf B Frommer  Is a corresponding author
  1. Carnegie Institution for Science, United States
8 figures

Figures

Design and development of NiTrac sensors.

(A) Schematic representation of the NiTrac1 sensor construct. Aphrodite, yellow; mCerulean, light blue; CHL1/NRT1.1/NPF6.3 dark blue; TM, transmembrane domain. (B) Emission spectra for NiTrac1 …

https://doi.org/10.7554/eLife.01917.003
PepTrac sensors.

(A) Schematic representation of the PepTrac sensor constructs. AtPTR1, 2, 4, and 5 were used for PepTrac sensor creation. Three-dimensional model of AFP-PTRs-mCerulean chimeric protein based on the …

https://doi.org/10.7554/eLife.01917.004
Biphasic kinetics of the NiTrac1 response.

(A) Biphasic nitrate uptake kinetics of the fluorescence response of NiTrac1 (red) and biphasic nitrate uptake transport kinetics of CHL1/NRT1.1 (Black). (B) Monophasic nitrate uptake kinetics of …

https://doi.org/10.7554/eLife.01917.005
Response of NiTrac1 mutants to nitrate addition.

(A) Three-dimensional model of CHL1 protein based on the crystal structures of bacteria (‘Materials and methods’). Red square, potential substrate binding pocket. Right panel, enlarged potential …

https://doi.org/10.7554/eLife.01917.006
Current and voltage curve of CHL1/NRT1.1 mutants using TEVC.

Oocytes were voltage clamped at −40 mV and stepped into a test voltage between −20 and −180 mV for 300 ms, in −20-mV increments. The currents (I) shown here are the difference between the currents …

https://doi.org/10.7554/eLife.01917.007
15NO3 uptake activity of various CHL1 mutants in oocytes.

The injected oocytes with various cRNA of CHL1 mutants were incubated with 0.25 mM and 10 mM K15NO3 buffer at pH 5.5 for about 1.5∼2 hr, and their 15N content was determined as described in …

https://doi.org/10.7554/eLife.01917.008
Summary of nitrate uptake and TEVC measurements for CHL1 mutants and relative fluorescence emission and change in apparent energy transfer efficiency of NiTrac1 mutants in presence and absence of nitrate.

CHL1 column: wild type and various mutants of CHL1. 15N column: nitrate uptake measured in oocytes using 15NO3 (checkmark indicates transport activity detected, whereas × indicates no …

https://doi.org/10.7554/eLife.01917.009
Effects of the fluorescence response of NiTrac1 by interacting proteins.

Known interactors or regulators, such as CIPK8, CIPK23, CBL1, and CBL9 as well as other interactors identified in a large-scale membrane protein interaction screen were co-expressed with NiTrac1 in …

https://doi.org/10.7554/eLife.01917.010

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