植物铜转运蛋白COPT1内吞和非经典分泌机制及其在铜稳态平衡中的作用

Copper (Cu) is an essential nutrition element for plant growth and development, however, deficient or excess copper is detrimental to plants. Therefore plants have evolved complex and elaborate homeostatic mechanisms to control intracellular copper concentration. High-affinity copper transporters have a vital role in regulating copper homeostasis, whereas the mechanisms of their endocytosis, secretion and intracellular transport are unknown. In this project,we will employ several new methods and techniques, encompassing biochemistry, molecular and celluar biology, imaging (High Content Imaging System) and colloidal gold immunoelectron microscopy.This project will focus on the following three aspects of research using Arabidopsis highe-affinity copper transporter 1 (COPT1) protein: 1) To observe distribution pattern and determine contents of COPT1 in plasma membrane before and after Cu induction; 2) To study endocytic pathway and intracellular transport mode of COPT1, and their effects on copper flux and copper accumulation; 3) To reveal mechanism of unconventional secretion of COPT1 and its role in copper homeostasis. These results will not only be significant to go deeply how plants adapt to variations of environmental Cu levels, but also provide potential applications for creating engineered plants by molecular design for phytoremediation of copper polluted environment.

铜(Cu)是植物生长发育所必需的营养元素，但Cu缺乏或过量均会对植物产生毒害效应。因而植物在长期的进化过程中形成了一套内稳态机制来调控细胞内铜离子浓度。高亲和铜转运蛋白在Cu离子稳态调节过程中发挥重要功能，然而这些蛋白的内吞、分泌及胞内转运机制尚不清楚。本项目拟以拟南芥高亲和铜转运蛋白COPT1为研究对象，应用细胞生物学、分子生物学、生物化学等综合分析手段，结合高内涵细胞成像系统和胶体金免疫电镜等新技术，开展如下研究：1）研究Cu刺激前后COPT1在质膜上的分布模式及含量变化；2）明确COPT1的内吞途径和胞内转运模式以及对Cu离子流和Cu积累的影响；3）揭示COPT1的非经典分泌机制及在细胞Cu离子稳态平衡中的作用。这不仅对深入探讨植物如何适应环境Cu水平变化具有重要理论意义，而且为通过分子设计创制用于Cu污染环境修复工程植物提供有应用价值的基础。

COPT (copper transporter)家族蛋白对Cu具有高亲和性和专一性，其家族中的COPT1定位于质膜，被认为对Cu的吸收起关键作用。本项目以COPT1为研究对象，对其在Cu离子稳态平衡中的作用机制进行研究。获得的主要研究结果包括：(1) 对突变体copt1-1、copt2-1和双突变体copt1-1copt2-1的表型观察后发现，Cu缺乏时，双突变体copt1-1copt2-1的生长受到严重抑制；Cu过量显著抑制COPT1转基因植株的生长。突变体copt1-1、copt2-1或copt2-2 的莲座叶中积累较少的Cu，而COPT1转基因植株的根和花中则积累较多的Cu。(2) 用50 μM Cu处理后，COPT1-GFP在质膜上的定位迅速减少。Western blot实验表明，随Cu处理时间的延长，COPT1-GFP蛋白水平逐渐降低。蛋白酶体抑制剂MG132能够抑制Cu诱导的COPT1-GFP蛋白水平的降低。在26S蛋白酶体亚基突变体 (rpn9a和rpn8b) 中也发现，Cu处理后COPT1-GFP蛋白水平降低不明显。Cu处理下突变体rpn9a和rpn8b生长明显受到抑制，且其体内积累高水平的Cu。（3）对COPT1 N端Cu结合结构域进行定点突变或缺失突变后的酵母功能互补实验显示，COPT1缺失N端序列后，转化酵母的表型不能恢复，而仅将Met44突变后，转化酵母的表型可以恢复，另外我们发现，His43对COPT1的Cu吸收功能至关重要。以上结果表明，COPT1在拟南芥对Cu的吸收中发挥重要作用，其N端结构域为它们正常行使功能所必需, 拟南芥可以通过调节COPT1在质膜上的丰度进而维持细胞的Cu离子稳态。