Data from: The clathrin adaptor complex AP-2 mediates endocytosis of BRASSINOSTEROID INSENSITIVE1 in Arabidopsis

Clathrin-mediated endocytosis (CME) regulates many aspects of plant development, including hormone signaling and responses to environmental stresses. Despite the importance of this process, the machinery that regulates CME in plants is largely unknown. In mammals, the heterotetrameric ADAPTOR PROTEIN COMPLEX-2 (AP-2) is required for the formation of clathrin-coated vesicles at the plasma membrane (PM). Although the existence of AP-2 has been predicted in Arabidopsis thaliana, the biochemistry and functionality of the complex is still uncharacterized. Here, we identified all the subunits of the Arabidopsis AP-2 by tandem affinity purification and found that one of the large AP-2 subunits, AP2A1, localized at the PM and interacted with clathrin. Furthermore, endocytosis of the leucine-rich repeat receptor kinase, BRASSINOSTEROID INSENSITIVE1 (BRI1), was shown to depend on AP-2. Knockdown of the two Arabidopsis AP2A genes or overexpression of a dominant-negative version of the medium AP-2 subunit, AP2M, impaired BRI1 endocytosis and enhanced the brassinosteroid signaling. Our data reveal that the CME machinery in Arabidopsis is evolutionarily conserved and that AP-2 functions in receptor-mediated endocytosis.网格蛋白介导的内吞作用（Clathrin-mediated endocytosis, CME）调控植物发育的诸多环节，包括激素信号转导与环境胁迫应答。尽管该过程至关重要，但植物体内调控CME的分子机制仍未明确。在哺乳动物中，异四聚体衔接蛋白复合物2（ADAPTOR PROTEIN COMPLEX-2, AP-2）是质膜（plasma membrane, PM）处网格蛋白包被囊泡形成所必需的。尽管学界已预测拟南芥（Arabidopsis thaliana）中存在AP-2复合物，但该复合物的生化特性与功能仍未被表征。本研究通过串联亲和纯化技术鉴定了拟南芥AP-2的全部亚基，并发现AP-2的大型亚基之一AP2A1定位于质膜，且可与网格蛋白相互作用。进一步研究表明，富含亮氨酸重复序列受体激酶BRASSINOSTEROID INSENSITIVE1（BRI1）的内吞作用依赖于AP-2。敲低拟南芥的两个AP2A基因，或过表达AP-2中型亚基AP2M的显性负突变体，均会削弱BRI1的内吞作用并增强油菜素内酯信号通路。本研究数据证实，拟南芥体内的CME机制具有进化保守性，且AP-2参与受体介导的内吞过程。