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.

中文翻译：

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CIPK15激酶介导的AMT1 NH ₄ ⁺转运蛋白的反馈抑制

铵（NH4 +）是一种关键的氮形式，当其积累到很高的水平时，会变得有毒。铵转运蛋白（AMT）是负责吸收NH4 +的关键转运蛋白。AMT活性受变构反馈控制，由胞质C端（CCT）中苏氨酸的磷酸化介导。然而，负责NH 4 +触发的磷酸化的激酶仍然未知。在这项研究中，功能筛选确定了蛋白激酶CBL相互作用蛋白激酶15（CIPK15）是AMT1; 1活性的负调节剂。CIPK15能够与质膜上的几个AMT1旁系同源物相互作用。分析AmTryoshka，一种用于酵母中AMT1; 3的NH4 +转运蛋白活性传感器，以及爪蟾卵母细胞中AMT1; 1的两电极电压钳（TEVC），表明CIPK15抑制了AMT活性。当幼苗暴露于升高的NH4 +水平时，CIPK15转录水平升高。值得注意的是，与野生型相比，cipk15基因敲除突变体显示出更高的15NH4 +吸收和积累的NH4 +量。一致地，cipk15对NH4 +和甲基铵均敏感，但对硝酸盐（NO3-）不敏感。两者合计，我们的数据表明AMT1活性的反馈抑制是由蛋白激酶CIPK15通过CCT中残基的磷酸化来介导，以减少NH4 +的积累。