Elements of the ERAD ubiquitin ligase Doa10 regulating sequential poly-ubiquitylation of its targets

In ER-associated degradation (ERAD), misfolded ER proteins are degraded by the proteasome after undergoing ubiquitylation. Yeast Doa10 (human MARCHF6/TEB4) is a membrane-embedded E3 ubiquitin ligase that functions with E2s Ubc6 and Ubc7. Ubc6 attaches a single ubiquitin to substrates, which is extended by Ubc7 to form a polyubiquitin chain. We show the conserved C-terminal element (CTE) of Doa10 promotes E3-mediated Ubc6 activity. Doa10 substrates undergoing an alternative ubiquitylation mechanism are still degraded in CTE-mutant cells. Structure prediction by AlphaFold2 suggests the CTE binds near the catalytic RING-CH domain, implying a direct role in substrate ubiquitylation, and we confirm this interaction using intragenic suppression. Truncation analysis defines a minimal E2-binding region of Doa10; structural predictions suggest that Doa10 forms a retrotranslocation channel and that E2s bind within the cofactor-binding region defined here. These results provide mechanistic insight into how Doa10, and potentially other ligases, interact with their cofactors and mediate ERAD.