biostudies-literatureWu KU.S. Department of Health & Human Services | NIH | National Institute of General Medical SciencesPew Charitable TrustsHellman FoundationVallee FoundationJane Coffin Childs Memorial Fund for Medical ResearchNIGMS NIH HHSNational Science Foundation2182https://www.ebi.ac.uk/biostudies/studies/S-EPMC10928120biostudies-literatureUnknown15(1)Doa10 (MARCHF6 in metazoans) is a large polytopic membrane-embedded E3 ubiquitin ligase in the endoplasmic reticulum (ER) that plays an important role in quality control of cytosolic and ER proteins. Although Doa10 is highly conserved across eukaryotes, it is not understood how Doa10 recognizes its substrates. Here, we define the substrate recognition mechanism of Doa10 by structural and functional analyses on Saccharomyces cerevisiae Doa10 and its model substrates. Cryo-EM analysis shows that Doa10 has unusual architecture with a large lipid-filled central cavity, and its conserved middle domain forms an additional water-filled lateral tunnel open to the cytosol. Our biochemical data and molecular dynamics simulations suggest that the entrance of the substrate's degron peptide into the laNature communicationsSubstrate recognition mechanism of the endoplasmic reticulum-associated ubiquitin ligase Doa10.PMC109281201828187R01 GM123169Wu KGumbart JCLynch DLItskanov SChen YTurner APark EfalseSubstrate recognition mechanism of the endoplasmic reticulum-associated ubiquitin ligase Doa10.Doa10 (MARCHF6 in metazoans) is a large polytopic membrane-embedded E3 ubiquitin ligase in the endoplasmic reticulum (ER) that plays an important role in quality control of cytosolic and ER proteins. Although Doa10 is highly conserved across eukaryotes, it is not understood how Doa10 recognizes its substrates. Here, we define the substrate recognition mechanism of Doa10 by structural and functional analyses on Saccharomyces cerevisiae Doa10 and its model substrates. Cryo-EM analysis shows that Doa10 has unusual architecture with a large lipid-filled central cavity, and its conserved middle domain forms an additional water-filled lateral tunnel open to the cytosol. Our biochemical data and molecular dynamics simulations suggest that the entrance of the substrate's degron peptide into the la2024-01-01T00:00:00Z2024 Mar2025-04-22T13:03:53.693Z2025-04-06T00:29:52.24ZS-EPMC109281203846763810.1038/s41467-024-46409-2