The plant trans-Golgi network (TGN) is a central trafficking hub where secretory, vacuolar, recycling, and endocytic pathways merge. Among currently known molecular players involved in TGN transport, three different adaptor protein (AP) complexes promote vesicle generation at the TGN with different cargo specificity and destination. Yet, it remains unresolved how sorting into diverging vesicular routes is spatially organized. Here, we study the family of Arabidopsis thaliana Epsin-like proteins, which are accessory proteins to APs facilitating vesicle biogenesis. By comprehensive molecular, cellular, and genetic analysis of the EPSIN gene family, we identify EPSIN1 and MODIFIED TRANSPORT TO THE VACUOLE1 (MTV1) as its only TGN-associated members. Despite their large phylogenetic distance, they perform overlapping functions in vacuolar and secretory transport. By probing their relationship with AP complexes, we find that they define two molecularly independent pathways: While EPSIN1 associates with AP-1, MTV1 interacts with AP-4, whose function is required for MTV1 recruitment. Although both EPSIN1/AP-1 and MTV1/AP-4 pairs reside at the TGN, high-resolution microscopy reveals them as spatially separate entities. Our results strongly support the hypothesis of molecularly, functionally, and spatially distinct subdomains of the plant TGN and suggest that functional redundancy can be achieved through parallelization of molecularly distinct but functionally overlapping pathways.

植物反式-高尔基体网络（TGN）是一个中央运输枢纽，分泌，液泡，循环利用和内吞途径在此融合。在目前涉及TGN运输的分子参与者中，三种不同的衔接蛋白（AP）复合物以不同的货物特异性和目的地促进TGN处的囊泡生成。然而，如何分类到不同的囊泡途径在空间上仍是未知的。在这里，我们研究拟南芥的家庭类脂蛋白样蛋白，是AP的辅助蛋白，可促进囊泡的生物发生。通过对EPSIN基因家族进行全面的分子，细胞和遗传分析，我们确定EPSIN1和到VACUOLE1（MTV1）的修饰运输是其唯一的TGN相关成员。尽管它们的系统发育距离很大，但它们在液泡和分泌转运中执行重叠功能。通过探究它们与AP复合物的关系，我们发现它们定义了两个分子独立的途径：EPSIN1与AP-1缔合，而MTV1与AP-4相互作用，AP-4的功能是招募MTV1所必需的。尽管EPSIN1 / AP-1和MTV1 / AP-4对都位于TGN，但高分辨率显微镜显示它们在空间上是分开的实体。我们的结果有力地支持了分子，功能，