Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent 9052, Belgium.
Plant Cell. 2022 May 24;34(6):2150-2173. doi: 10.1093/plcell/koac071.
In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.
在真核生物中，网格蛋白包被小泡（CCVs）促进了细胞表面物质的内化以及高尔基体后运输途径中货物的运动。这种功能多样性部分由多种单体和多聚网格蛋白衔接子复合物提供，这些复合物提供了区室和货物的选择性。衔接蛋白组装多肽-1（AP-1）复合物在跨高尔基网络（TGN）处作为分泌途径的一部分发挥作用，而 AP-2 复合物和 TPLATE 复合物则共同在质膜处发挥作用，以执行网格蛋白介导的内吞作用。深入了解植物中的网格蛋白介导运输的关键将是全面鉴定和表征参与 CCV 形成和靶向的进化保守和植物特异性核心和辅助机制的网络。为了促进这些研究，我们分析了从分裂和扩展悬浮培养的拟南芥（Arabidopsis thaliana）细胞中分离的富含 TGN/早期内体衍生和内吞 CCV 的蛋白质组。串联质谱分析结果通过差异化学标记实验进行了验证，以鉴定与 CCV 共同富集的蛋白质。在 CCV 中富集的蛋白质包括先前表征的 CCV 成分和货物，如液泡分选受体，以及保守和植物特异性成分，其在网格蛋白介导的运输中的功能尚未被定义。值得注意的是，除了 AP-1 和 AP-2，AP-4 复合物的所有亚基都在 CCV 蛋白质组中高度丰富，但 AP-3 或 AP-5 则不然。AP-4 与悬浮培养的拟南芥 CCV 的关联通过其他生化数据得到进一步支持。
