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bioRxiv · 10.1101/2020.11.03.367383

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

Chen, H.-Y.·Chen, Y.-N.·Wang, H.-Y.·Liu, Z.-T.·Frommer, W. B.·Ho, C.-H.
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Abstract

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). However, the kinases responsible for the NH4+-triggered phosphorylation remain unknown. In 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-). Taken 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.

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Chen, H.-Y., Chen, Y.-N., Wang, H.-Y., Liu, Z.-T., Frommer, W. B., Ho, C.-H.. 2020-11-04. Feedback inhibition of AMT1 NH4+-transporters mediated by CIPK15 kinase. https://doi.org/10.1101/2020.11.03.367383

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