Transmembrane E3 ligases play crucial roles in homeostasis. Much protein and organelle quality control, and metabolic regulation, are determined by ER-resident MARCH6 E3 ligases, including Doa10 in yeast. Here, we present Doa10/MARCH6 structural analysis by cryo-EM and AlphaFold predictions, and a structure-based mutagenesis campaign. The majority of Doa10/MARCH6 adopts a unique circular structure within the membrane. This channel is established by a lipid-binding scaffold, and gated by a flexible helical bundle. The ubiquitylation active site is positioned over the channel by connections between the cytosolic E3 ligase RING domain and the membrane-spanning scaffold and gate. Here, by assaying 95 MARCH6 variants for effects on stability of the well-characterized substrate SQLE, which regulates cholesterol levels, we reveal crucial roles of the gated channel and RING domain consistent with AlphaFold-models of substrate-engaged and ubiquitylation complexes. SQLE degradation further depends on connections between the channel and RING domain, and lipid binding sites, revealing how interconnected Doa10/ MARCH6 elements could orchestrate metabolic signals, substrate binding, and E3 ligase activity.

48 h post-transduction, GFP/mCherry-positive cells were sorted on a BC CytoFLEX SRT (Beckman Coulter)

Bottom: Flow cytometry panels comparing SQLE reporter levels in MARCH6-depleted cells re-expressing MARCH6WT, MARCH6ΔRING, or four MARCH6 variants with mutations outlined above (top). Relative mCherry fluorescence normalized to GFP as an expression control presented as a histogram. Representative result shown from six independent biological replicates.

d. Western blot data