Plants have evolved sophisticated mechanisms to adjust to deficiency or excess of nutrients. Membrane transport proteins play a central role in nutrient uptake from soil. In Arabidopsis thaliana, the COPPER TRANSPORTOR (COPT) family encodes high-affinity copper transporters. COPT2 is transcriptionally regulated in response to changing levels of cellular copper. However, little is known about whether COPT2 activity is subject to multiple levels of regulation. Here, we showed that the plasma membrane-/endoplasmic reticulum-resident COPT2 protein is degraded in response to excess copper. Confocal microscopy analysis together with pharmacological treatment with a vesicle trafficking inhibitor or vacuolar ATPase inhibitor indicated that copper-mediated downregulation of COPT2 is unlikely to be controlled by endosomal recycling and vacuolar system. However, COPT2 protein is stabilized by proteasome inhibition. Through site-directed mutagenesis, we found that COPT2 cannot be ubiquitinated, and lysine residues at the C-terminus is dispensable for copper-induced degradation of COPT2 but required for copper acquisition. Altogether, our findings reveal that unlike many metal transporters in Arabidopsis, COPT2 is a substrate of ubiquitin-independent proteasomal degradation but not of vacuolar proteases. These findings highlight the mechanistic diversity and complexity of plasma membrane transporter degradation.

植物已经进化出复杂的机制来适应营养素的缺乏或过量。膜转运蛋白在土壤养分吸收中起着核心作用。在拟南芥中，COPPER TRANSPORTOR（COPT）系列编码高亲和力铜转运蛋白。COPT2受细胞铜水平变化的转录调控。但是，关于COPT2活性是否受多种调控的了解甚少。在这里，我们表明，质膜/内质网驻留的COPT2蛋白响应过量的铜而降解。共聚焦显微镜分析以及用囊泡运输抑制剂或液泡ATPase抑制剂进行的药理处理表明，铜介导的COPT2下调不太可能由内体再循环和液泡系统控制。但是，COPT2蛋白通过蛋白酶体抑制作用得以稳定。通过定点诱变，我们发现COPT2不能泛素化，C端的赖氨酸残基对于铜诱导的COPT2降解是必不可少的，但对于铜的获取却是必需的。总之，我们的发现表明，与拟南芥中的许多金属转运蛋白不同，COPT2是不依赖泛素的蛋白酶体降解的底物，而不是液泡状蛋白酶的底物。这些发现突出了质膜转运蛋白降解的机制多样性和复杂性。