Feedback inhibition of AMT1 NH 4 + -transporters mediated by CIPK15 kinase
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RESEARCH ARTICLE
Open Access
Feedback inhibition of AMT1 NH4+transporters mediated by CIPK15 kinase Hui-Yu Chen1, Yen-Ning Chen1, Hung-Yu Wang1, Zong-Ta Liu1, Wolf B. Frommer2,3 and Cheng-Hsun Ho1*
Abstract Background: 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. Results: 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-electrodevoltage-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−). Conclusion: 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. Keywords: Arabidopsis thaliana, Ammonium, Protein kinase, Phosphorylation, Transporter
Background As a key building block of nucleic acids, amino acids, and proteins, nitrogen is an essential nutrient. In plants, nitrogen supply can limit or inhibit growth, development, and crop yield when below or above the optimal range. Ammonium (NH4+) is one of the main inorganic forms of nitrogen for plant nutrition. NH4+ is also an important nitrogen source for bacteria, fungi, and plants, but becomes toxic when it rises above certain levels [1–5]. Plants take up NH4+ with the help of specific transporters. AMT/MEP/Rhesus protein superfamily members function as electrogenic high-affinity NH4+ transporters [6–9]. Potassium (K+) channels can also mediate NH4+ uptake [10]. The Arabidopsis genome contains six AMT * Correspondence: [email protected] 1 Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan Full list of author information is available at the end of the article
paralogs, four (AMT1;1, 1;2, 1;3, and 1;5) of which are together essential for NH4+ uptake [11, 12]. Unlike K+ channels, AMTs are highly selective for NH4+ and its methylated form, methylammonium (MeA) [6, 13]. In addition to their roles as transporters, AMTs can also function as receptors involved in the control of root growth and development, similar to the yeast MEP2 transceptor, which measures NH4+ concentrations to regulate pseudohyphal growth [14]. Recently, a ratiometric biosensor of NH4+ transporter ac
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