Cellular membrane homoeostasis is maintained via a tightly regulated membrane and cargo flow between organelles of the endocytic and secretory pathways. Adaptor protein complexes (APs), which are recruited to membranes by the small GTPase ARF1, facilitate cargo selection and incorporation into trafficking intermediates. According to the classical model, small vesicles would facilitate bi-directional long-range transport between the Golgi, endosomes and plasma membrane. Here we revisit the intracellular organization of the vesicular transport machinery using a combination of CRISPR-Cas9 gene editing, live-cell high temporal (fast confocal) or spatial (stimulated emission depletion) microscopy as well as correlative light and electron microscopy. We characterize tubulo-vesicular ARF1 compartments that harbour clathrin and different APs. Our findings reveal two functionally different classes of ARF1 compartments, each decorated by a different combination of APs. Perinuclear ARF1 compartments facilitate Golgi export of secretory cargo, while peripheral ARF1 compartments are involved in endocytic recycling downstream of early endosomes. Contrary to the classical model of long-range vesicle shuttling, we observe that ARF1 compartments shed ARF1 and mature into recycling endosomes. This maturation process is impaired in the absence of AP-1 and results in trafficking defects. Collectively, these data highlight a crucial role for ARF1 compartments in post-Golgi sorting.

细胞膜匀态通过内吞途径和分泌途径的细胞器之间的严格调节膜和货物流来维持。由小 GTP 酶 ARF1 募集到膜上的接头蛋白复合物 （AP） 有助于货物选择和掺入运输中间体。根据经典模型，小囊泡将促进高尔基体、内体和质膜之间的双向远程运输。在这里，我们结合使用 CRISPR-Cas9 基因编辑、活细胞高时间（快速共聚焦）或空间（受激发射耗竭）显微镜以及相关光学和电子显微镜，重新审视了囊泡运输机制的细胞内组织。我们表征了含有网格蛋白和不同 AP 的管泡 ARF1 隔室。我们的研究结果揭示了两类功能不同的 ARF1 隔室，每个隔室都由不同的 AP 组合装饰。核周 ARF1 隔室促进高尔基体分泌物的出口，而外周 ARF1 隔室参与早期内体下游的内吞循环。与长程囊泡穿梭的经典模型相反，我们观察到 ARF1 区室脱落 ARF1 并成熟为回收内体。在没有 AP-1 的情况下，这种成熟过程会受到影响，并导致贩运缺陷。总的来说，这些数据突出了 ARF1 区室在后高尔基体分选中的关键作用。