Fig. 4: Membrane recruitment of the AP-4 core mediated by ARF1.

a Structural alignments of the two AP-4/ARF1 complexes with a membrane-docked ARF1 to construct a membrane-anchored AP-4/ARF1 complex model. In two structural models, the structure of GTP-bound ARF1 (PDB: 2KSQ) is aligned with ARF1 in either the μ4-CTD-docked (left panel) or μ4-CTD-undocked (right panel) AP-4/ARF1 complex. The μ4-CTD/APP-peptide complex structure (PDB: 3l81) is shown in the middle panel, with the cargo-binding pocket highlighted. b Structural visualization of the AP-4-ε/ARF1 interface derived from the μ4-CTD-docked, μ4-CTD-undocked AP-4/ARF1 complex or AlphaFold3-predicted AP-4-ε/ARF1 model. The interface-related helix 3 (α3) and helix 5 (α5) in AP-4-ε are labeled. c, d A combined ribbon-and-stick model showing the interface between AP-4-ε and ARF1 in the μ4-CTD-docked (c) or μ4-CTD-undocked (d) AP-4/ARF1 complex. The Switch Ⅰ and Switch Ⅱ regions of ARF1 are labeled. e Strep-pull-down assay of ARF1 and the AP-4 core complex (wild-type and mutants). Protein samples were analyzed by SDS-PAGE and immunoblotting. The experiment is independently repeated three times (n = 3). Source data are provided as a Source Data file. f A schematic diagram of the liposome flotation assay used to detect ARF1-mediated membrane recruitment of the AP-4 core. After centrifuging, LUV (pink), streptavidin or streptavidin-ARF1 (green) and/or the AP-4 core complex (WT or L133E mutant, orange) were co-floated on top of the density gradients, and remaining unbounded proteins left at the bottom. g, k Flotation analyses using LUVs (69% DOPC, 5% DOPS, 10% DOPE, 14% Choline, 1% Rhodamine-PE and 1% Biotinyl Cap PE) incubated with (g) streptavidin-ARF1; (h) AP-4 core-WT; (i) streptavidin + AP-4 core-WT; (j) streptavidin-ARF1 + AP-4 core-WT; (k) streptavidin-ARF1 + AP-4 core-L133E. Each experiment is independently repeated three times (n = 3). Source data are provided as a Source Data file.