Abstract：
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.
摘要：
铜的获取和随后向靶蛋白的递送对于许多生物过程是必需的。然而,这种微量元素的细胞水平必须控制，因为它的潜在毒性。富含潜在金属结合氨基酸的COPT1蛋白在拟南芥细胞质膜的高亲和力铜吸收中起作用。这些推定的金属结合残基的功能作用在很大程度上是未知的。通过截短和定点突变,我们确定His43是细胞外N末端结构域内的一个单一残基,对于COPT1的铜摄取是绝对关键的.用亮氨酸取代这个残基，甲硫氨酸或半胱氨酸几乎使COPT1的转运功能失活，这意味着His43在COPT1活性的调节中不能作为铜配体。所有细胞外N末端金属结合残基的缺失完全阻断了铜刺激的降解，但不会改变COPT1的亚细胞分布和多聚化。尽管His43突变为丙氨酸和丝氨酸保留了酵母细胞中的转运蛋白活性，突变蛋白在拟南芥细胞的蛋白酶体中不稳定并被降解。我们的结果证明了细胞外残基His43在高亲和力铜转运活性中的关键作用，并提出了调节COPT1金属转运和蛋白质稳定性的共同分子机制。
