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bioRxiv · 10.1101/2023.10.27.564143

Multi-functional ARF1 compartments serve as a hub for short-range cargo transfer to endosomes

Stockhammer, A.·Adarska, P.·Natalia, V.·Heuhsen, A.·Klemt, A.·Harel, S.·Rodilla-Ramirez, C.·Spalt, C.·Oezsoy, E.·Leupold, P.·Grindel, A.·Fox, E.·Mejedo, J. O.·Puchkov, D.·Haucke, V.·Bottanelli, F.
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Abstract

Cellular membrane homeostasis 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 (STED)) microscopy as well as correlative light and electron microscopy. We characterize novel tubulo-vesicular ARF1 compartments that harbor 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.

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Stockhammer, A., Adarska, P., Natalia, V., Heuhsen, A., Klemt, A., Harel, S., Rodilla-Ramirez, C., Spalt, C., Oezsoy, E., Leupold, P., Grindel, A., Fox, E., Mejedo, J. O., Puchkov, D., Haucke, V., Bottanelli, F.. 2023-10-27. Multi-functional ARF1 compartments serve as a hub for short-range cargo transfer to endosomes. https://doi.org/10.1101/2023.10.27.564143

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