Feedback inhibition of AMT1 NH4+-transporters mediated by CIPK15 kinase

Hui-Yu Chen,Zong-Ta Liu,Hung-Yu Wang,Cheng-Hsun Ho,Yen-Ning Chen,Wolf B Frommer

doi:10.1186/s12915-020-00934-w

Abstract

BackgroundAmmonium (NH4+), a key nitrogen form, becomes toxic when it accumulates to high levels. Ammonium transporters (AMTs) are the key transporters responsible for NH4+ uptake. AMT activity is under allosteric feedback control, mediated by phosphorylation of a threonine in the cytosolic C-terminus (CCT). However, the kinases responsible for the NH4+-triggered phosphorylation remain unknown.ResultsIn this study, a functional screen identified protein kinase CBL-Interacting Protein Kinase15 (CIPK15) as a negative regulator of AMT1;1 activity. CIPK15 was able to interact with several AMT1 paralogs at the plasma membrane. Analysis of AmTryoshka, an NH4+ transporter activity sensor for AMT1;3 in yeast, and a two-electrode-voltage-clamp (TEVC) of AMT1;1 in Xenopus oocytes showed that CIPK15 inhibits AMT activity. CIPK15 transcript levels increased when seedlings were exposed to elevated NH4+ levels. Notably, cipk15 knockout mutants showed higher 15NH4+ uptake and accumulated higher amounts of NH4+ compared to the wild-type. Consistently, cipk15 was hypersensitive to both NH4+ and methylammonium but not nitrate (NO3−).ConclusionTaken together, our data indicate that feedback inhibition of AMT1 activity is mediated by the protein kinase CIPK15 via phosphorylation of residues in the CCT to reduce NH4+-accumulation.

Highlights

Ammonium (NH4+), a key nitrogen form, becomes toxic when it accumulates to high levels
NH4+-induced inward currents were completely blocked by a mixture of CIPK2, 10, 15, 20, and 26, while the combination of CIPK3, 8, 9, 23, and 24 had no major impact on AMT1;1 activity (Additional file 1: Figure S1)
Because we focused on Calcineurin B-like protein (CBL)-Interacting Protein Kinase15 (CIPK15), we cannot exclude the possibility that other CIPKs may affect Ammonium transporters (AMTs) activity, in particular when co-expressed with CBLs

Summary

Introduction

Ammonium (NH4+), a key nitrogen form, becomes toxic when it accumulates to high levels. Ammonium transporters (AMTs) are the key transporters responsible for NH4+ uptake. AMT activity is under allosteric feedback control, mediated by phosphorylation of a threonine in the cytosolic C-terminus (CCT). As a key building block of nucleic acids, amino acids, and proteins, nitrogen is an essential nutrient. Ammonium (NH4+) is one of the main inorganic forms of nitrogen for plant nutrition. NH4+ is an important nitrogen source for bacteria, fungi, and plants, but becomes toxic when it rises above certain levels [1,2,3,4,5]. AMT/MEP/Rhesus protein superfamily members function as electrogenic high-affinity NH4+ transporters [6,7,8,9].

Results

Discussion

Conclusion