Ectopic RING activity at the ER membrane differentially impacts ERAD protein quality control pathways

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control pathway that ensures misfolded pro-teins are removed from the ER and destroyed. In ERAD, membrane and luminal substrates are ubiquitylated by ER-resident RING-type E3 ubiquitin ligases, retrotranslocated into the cytosol, and degraded by the proteasome. Over-expression of ERAD factors is frequently used in yeast and mammalian cells to study this process. Here, we analyze the impact of ERAD E3 overexpression on substrate turnover in yeast, where there are three ERAD E3 complexes (Doa10, Hrd1, and Asi1-3). Elevated Doa10 or Hrd1 (but not Asi1) RING ac-tivity at the ER membrane resulting from protein over-expression inhibits the degradation of specific Doa10 substrates. The ERAD E2 ubiquitin-conjugating enzyme Ubc6 becomes limiting under these conditions, and UBC6 over-expression restores Ubc6-mediated ERAD. Using a subset of the dominant-negative mutants, which contain the Doa10 RING domain but lack the E2-binding region, we show that they induce degradation of membrane tail-anchored Ubc6 independently of endogenous Doa10 and the other ERAD E3 complexes. This remains true even if the cells lack the Dfm1 rhomboid pseudoprotease, which is also a proposed retro-translocon. Hence, rogue RING activity at the ER membrane elicits a highly specific off-pathway defect in the Doa10 pathway, and the data point to an additional ERAD E3-independent retrotranslocation mechanism.

Automated summary

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control pathway that removes misfolded proteins from the ER. Overexpression of ERAD factors inhibits the degradation of specific substrates. This study reveals a potential retrotranslocation mechanism independent of ERAD E3 complexes.