Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, Tianjin 300387, China.
Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, Tianjin 300387, China.
Int J Biol Macromol. 2023 Jun 30;241:124404. doi: 10.1016/j.ijbiomac.2023.124404. Epub 2023 Apr 11.
Copper acquisition and subsequent delivery to target proteins are essential for many biological processes. However, the cellular levels of this trace element must be controlled because of its potential toxicity. The COPT1 protein rich in potential metal-binding amino acids functions in high affinity copper uptake at the plasma membrane of Arabidopsis cells. The functional role of these putative metal-binding residues is largely unknown. Through truncations and site-directed mutagenesis, we identified His43, a single residue within the extracellular N-terminal domain as absolutely critical for copper uptake of COPT1. Substitution of this residue with leucine, methionine or cysteine almost inactivated transport function of COPT1, implying that His43 fails to serves as a copper ligand in the regulation of COPT1 activity. Deletion of all extracellular N-terminal metal-binding residues completely blocked copper-stimulated degradation but did not alter the subcellular distribution and multimerization of COPT1. Although mutation of His43 to alanine and serine retained the transporter activity in yeast cells, the mutant protein was unstable and degraded in the proteasome in Arabidopsis cells. Our results demonstrate a pivotal role for the extracellular residue His43 in high affinity copper transport activity, and suggest common molecular mechanisms for regulating both metal transport and protein stability of COPT1.
铜的获取及其随后向靶蛋白的输送对许多生物过程都是至关重要的。然而，由于其潜在的毒性，必须控制细胞内的这种微量元素水平。富含潜在金属结合氨基酸的 COPT1 蛋白在拟南芥细胞的质膜上发挥高亲和力铜摄取的功能。这些假定的金属结合残基的功能在很大程度上是未知的。通过截断和定点突变，我们鉴定出胞外 N 端结构域中的单个残基 His43 对 COPT1 的铜摄取绝对至关重要。用亮氨酸、蛋氨酸或半胱氨酸替换该残基几乎使 COPT1 的转运功能失活，这表明 His43 在调节 COPT1 活性中不能作为铜配体。删除所有胞外 N 端金属结合残基完全阻止了铜刺激的降解，但并未改变 COPT1 的亚细胞分布和多聚化。尽管将 His43 突变为丙氨酸和丝氨酸保留了酵母细胞中的转运体活性，但突变蛋白在拟南芥细胞的蛋白酶体中不稳定并降解。我们的结果表明胞外残基 His43 在高亲和力铜转运活性中起着关键作用，并表明 COPT1 的金属转运和蛋白稳定性的调节具有共同的分子机制。
