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 2 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.
Kaufman et al. (Fri,) studied this question.