The adaptor protein (AP) complexes not only form the inner layer of clathrin coats but also have clathrin-independent roles in membrane traffic whose mechanisms are unknown. HIV-1 Nef hijacks AP-1 to sequester major histocompatibility complex class I (MHC-I), evading immune detection. We found that AP-1:Arf1:Nef:MHC-I forms a coat on tubulated membranes without clathrin and determined its structure. The coat assembles via Arf1 dimer interfaces. AP-1–positive tubules are enriched in cells upon clathrin knockdown. Nef localizes preferentially to AP-1 tubules in cells, explaining how Nef sequesters MHC-I. Coat contact residues are conserved across Arf isoforms and the Arf-dependent AP complexes AP-1, AP-3, and AP-4. Thus, AP complexes can self-assemble with Arf1 into tubular coats without clathrin or other scaffolding factors. The AP-1:Arf1 coat defines the structural basis of a broader class of tubulovesicular membrane coats as an intermediate in clathrin vesicle formation from internal membranes and as an MHC-I sequestration mechanism in HIV-1 infection.

Live-cell imaging was conducted on a Zeiss LSM900 with Airyscan 2.0 detection in SR-2Y mode, using the Zen Blue software. All imaging was conducted in an incubation chamber at 37°C and 5% CO₂. 10um Z-stack movies with 140nm intervals were acquired for all analyzed cells. During multi-channel imaging, all channels were frame-sequentially acquired before moving to the next Z-slice to minimize crosstalk and time offset. Images were batch processed by ZEN Blue software for Airyscan reconstruction using filter strength setting 5 and 3D processing.

To test for colocalization between Nef and AP1 signal, both channels from the same slice in a Z-stack was analyzed. Both channels were first background corrected using the 15 pixel median filtering as described above, and thresholded using the “Auto Threshold” Otsu and Li thresholds for the AP1 and Nef channels respectively in imageJ. Particles less than 0.10 μm² were filtered out. AP1 signal was then classified as “spheroid” if particles had circularity between 0.75 – 1.00 or as “tubule” if particles had circularity between 0 – 0.75 according to the “Analyze Particles” function. Using the thresholded images of AP1 and Nef, the Manders correlation coefficient was calculated for each type of AP1 structure using the JACoP plugin in imageJ. Specifically, the fraction of AP1 pixels overlapping with Nef pixels was reported for colocalization. To confirm that colocalization of AP1 and Nef was not random, the Van Steensel CCF function was generated using JACoP as well.

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