Architecture of clathrin-independent AP3:ARF1-coated carriers Kaufman, Jonathan G.G. Tagiltsev, Grigory Stalder, Danièle S. Taylor, Rebecca J. Sava, Ioana Guo, Hui Ciazynska, Katarzyna A. Zaccai, Nathan R. Gray, Sally R. Vallis, Yvonne ; https://orcid.org/0000-0002-5408-5906 Höning, Stefan Kelly, Bernard T. Gershlick, David C. Briggs, John A.G. Owen, David J. ddc:570 The AP3 complex mediates cargo sorting and carrier assembly for the trafficking of transmembrane proteins from endosomes to lysosomes. AP3 is generally believed to localize to clathrin-free, ARF1-positive, elongated carriers in cells, but the architecture of AP3-based coats was unknown. Using in vitro reconstitution and cryo–electron tomography, we demonstrate that AP3:ARF1 spontaneously remodels membranes containing cargo and the phosphoinositide PI(3,5)P <jats:sub>2</jats:sub> into tubular structures coated in spiraling rows of AP3 arches and ARF1 dimers. Targeted point mutations disrupting critical AP3:ARF1 and AP3:AP3 lattice interfaces disrupt AP3 recruitment, carrier formation, and lysosomal cargo trafficking in cells. We propose that AP3 generates tubular carriers on endosomes by organizing ARF1 dimers into elongated membrane-deforming arrays while simultaneously selecting cargo. By demonstrating that AP3:ARF1 can generate carriers without using a clathrin lattice, we explain the clathrin independence of AP3-mediated trafficking. American Association for the Advancement of Science 2026 info:eu-repo/semantics/article doc-type:article text http://purl.org/coar/resource_type/c_2df8fbb1 https://research-explorer.ista.ac.at/record/22306 https://research-explorer.ista.ac.at/download/22306/22331 Kaufman JGG, Tagiltsev G, Stalder DS, et al. Architecture of clathrin-independent AP3:ARF1-coated carriers. <i>Science Advances</i>. 2026;12(20). doi:<a href="https://doi.org/10.1126/sciadv.aed1529">10.1126/sciadv.aed1529</a> eng info:eu-repo/semantics/altIdentifier/doi/10.1126/sciadv.aed1529 info:eu-repo/semantics/altIdentifier/e-issn/2375-2548 https://creativecommons.org/licenses/by/4.0/ info:eu-repo/semantics/openAccess