Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Monday Speaker Abstracts

SELF-ASSEMBLY AND STRUCTURE OF A CLATHRIN-INDEPENDENT AP-1:ARF1 TUBULAR MEMBRANE COAT

Rick Hooy 1,2 ; Yuichiro Iwamoto 1,2 ; Dan A Tudorica 2,3 ; Xuefeng Ren 1,2 ; James H Hurley 1,2,4 ; 1 University of California, Berkeley, Molecular and Cell Biology, Berkeley, CA, USA 2 California Institute for Quantitative Biosciences, University of California, Berkeley , Berkeley, CA, USA 3 Graduate Group in Biophysics, University of California, Berkeley, Berkeley, CA, USA 4 Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA, USA

The adaptor protein (AP) complexes not only form the inner layer of clathrin coats but also have clathrin-independent roles in membrane traffic. How APs assemble on membranes to achieve diverse cargo trafficking functions (e.g. with and without clathrin) remains ill defined. Pathogens co-opt AP assemblies to (de)regulate trafficking of select cargo molecules; HIV-1 Nef hijacks AP-1 and its cofactor, Arf1, to sequester major histocompatibility complex class I (MHC-I) to evade immune detection. To determine how Nef hijacks AP-1:Arf1 cargo trafficking assemblies, we reconstituted Nef-mediated MHC-I downregulation in vitro and solved the subnanometer resolution structures of membrane-associated AP-1 assemblies by cryo-electron tomography and subtomogram averaging. We found that AP-1:Arf1:Nef:MHC-I forms a coat on tubulated membranes without clathrin. The coat assembles via Arf1 dimer interfaces. The stoichiometry of AP-1-to-Arf1 correlates with membrane tube diameter. In cells, AP-1–positive tubules are enriched 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 homologs 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.

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