To achieve membrane homeostasis, eukaryotic cells employ a complex protein machinery that orchestrates cargo flow between the Golgi apparatus, the plasma membrane and different classes of endosomes. Adaptor proteins facilitate selection, enrichment, and transport of various secretory and endocytic cargoes. Post-Golgi adaptor protein complexes (APs) as well as Golgi-localized, gamma-ear containing, ADP-ribosylation factor binding proteins (GGAs) are recruited to membranes by the small GTPase ARF1. Once on the membrane they bind cargoes and accessory factors, and according to classical models, drive the formation of small clathrin-coated vesicular transport intermediates to promote bidirectional transport between the Golgi and endosomes. The recent discovery of tubular, ARF1-positve transport intermediates decorated by clathrin, made us rethink the classical long-range vesicular model and brought us to investigate the function of these compartments in post-Golgi trafficking. In this work, I re-evaluated the localization and the function of different post-Golgi adaptor proteins in the context of tubular ARF1-transport intermediates. By combining CRISPR-Cas9 gene editing with interactome mapping and advanced imaging techniques, including live-cell confocal and stimulated emission depletion (STED) microscopy as well as correlative lightelectron microscopy, I discovered that ARF1 compartments are a novel class of tubulovesicular endosomal compartments. Importantly, the majority of non-endocytic clathrin and different adaptors including AP-1, AP-3 and GGAs localize to segregated nanodomains on ARF1 compartments. Settling a debate in the field, I observed that clathrin is only recruited to AP-1- but not AP-3-nanodomains. AP-1-nanodomains are found at the interface of ARF1 compartments and recycling endosomes and AP-1 knock-out (KO) causes the formation of long aberrant tubules possessing the identity of both compartments and disrupts cargo flow. These findings suggest that AP-1 mediates short-range transport from ARF1 compartments to recycling endosomes, possibly via a kiss-and-run mechanism rather than promoting longrange vesicular transport. Furthermore, I observe transient interaction of AP-1- and GGAnanodomains on ARF1 compartments and interactome analysis shows that AP-1 binds most of its cargoes only in the absence of GGAs. Thus, GGAs could act as switch in AP-1-mediated cargo sorting by regulating cargo-AP-1 interaction. Taken together, in this work I re-envision the mechanisms of adaptor-mediated cargo-transport and expand our understanding of how proteins are transferred between organelles. AP-1mediated cargo hand-over from newly described ARF1 compartments to recycling endosomes, redefines the role for AP-1 in post-Golgi transport. My data and recent literature suggest that post-Golgi communication occurs via transient interactions between compartments of a tubulovesicular endosomal network. In addition, I present an alternative model for the role of GGAs in AP-1-mediated protein sorting, where GGAs control which cargoes are sorted by AP-1.
