A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matα2 repressor degradation

TLDR

Substrate discrimination in the ubiquitin–proteasome system is governed by specific E3–E2 combinations, and the human ortholog of yeast DOA10 lies in the cri‑du‑chat syndrome critical region, implying that impaired ubiquitin ligation could contribute to this disorder. We identify Doa10/Ssm4 as an ER/nuclear envelope‑resident E3 that targets the soluble transcription factor Matα2 via its Deg1 degradation signal, possesses an active RING finger, works with E2s Ubc6 and Ubc7 to ubiquitinate Deg1 substrates, is required for degradation of at least one ER membrane protein, and, together with Hrd1, cooperatively eliminates aberrant ER proteins, as evidenced by increased cadmium sensitivity and constitutive unfolded protein response in a doa10Δ hrd1Δ double mutant.

Abstract

Substrate discrimination in the ubiquitin–proteasome system is believed to be dictated by specific combinations of ubiquitin–protein ligases (E3s) and ubiquitin-conjugating enzymes (E2s). Here we identify Doa10/Ssm4 as a yeast E3 that is embedded in the endoplasmic reticulum (ER)/nuclear envelope yet can target the soluble transcription factor Matα2. Doa10 contains an unusual RING finger, which has ubiquitin-ligase activity in vitro and is essential in vivo for degradation of α2 via its Deg1 degradation signal. Doa10 functions with two E2s, Ubc6 and Ubc7, to ubiquitinate Deg1 -bearing substrates, and it is also required for the degradation of at least one ER membrane protein. Interestingly, different short-lived ER proteins show distinct requirements for Doa10 and another ER-localized E3, Hrd1. Nevertheless, the two E3s overlap in function: A doa10 Δ hrd1 Δ mutant is far more sensitive to cadmium relative to either single mutant and displays strong constitutive induction of the unfolded protein response; this suggests a role for both E3s in eliminating aberrant ER proteins. The likely human ortholog of DOA10 is in the cri-du-chat syndrome critical region on chromosome 5p, suggesting that defective ubiquitin ligation might contribute to this common genetic disorder.