Deep mutational scanning of Saccharomyces cerevisiae Hrd1 ubiquitin ligase determining residues required for endoplasmic reticulum associated degradation function
We performed deep mutation scanning of the Saccharomyces cerevisiae Hrd1 ubiquitin ligase to measure the effect of all amino-acid mutations on the ability of Hrd1 to degrade substrates via endoplasmic reticulum associated degradation. Yeast cells expressing mutant Hrd1 libraries (Input) and fluorescently tagged substrates (a misfolded membrane protein and misfolded lumenal protein) were sorted using fluorescence-activated cell sorting to isolate wildtype-like Hrd1 (WT) or Hrd1 mutations inhibiting lumenal substrate degradation specifically (LDead).