Fig. 1.

NPL4-dependent ubiquitinated ER protein degradation. (a) Simplified model of ERAD. Unfolded and/or damaged ER proteins (a membrane protein is depicted, soluble lumenal proteins are also subject to ERAD) are ubiquitinated by the E2 ubiquitin-conjugating enzyme Ubc7p (recruited to the ER membrane by Cue1p) and the E3 ubiquitin ligases Hrd1p and Doa10p. The E2s Ubc6p and Ubc1p also make minor contributions to ER protein ubiquitination. Once ubiquitinated, an ERAD substrate requires the activity of the Npl4p–Ufd1p–Cdc48p chaperone to be fully extracted from the membrane and degraded by the proteasome. (b) Accumulation of ubiquitinated proteins in npl4-1 membranes. WT and npl4-1 cells expressing 6×His–Myc-tagged ubiquitin were subjected to subcellular fractionation. Equal amounts of total protein (10 μg) from total cell extract (lanes 1 and 2), soluble (lanes 3 and 4), and membrane (lanes 5 and 6) fractions were separated by SDS/PAGE and immunoblotted with anti-Myc antibodies. (c) Extragenic suppression of npl4-1 by deletion of genes encoding ERAD ubiquitination machinery. Yeast strains of the indicated genetic backgrounds were serially diluted, spotted onto rich-media plates, and incubated for 2–3 days at the indicated temperatures. The number of cells spotted in each dilution is indicated on the bottom. (d) Loss of UBC7 abrogates accumulation of ubiquitinated proteins in npl4-1 membranes. WT and npl4-1 cells ±Δubc7 expressing 6×His–Myc-tagged ubiquitin were subjected to subcellular fractionation. Equal amounts of total protein (10 μg) from total cell extract (lanes 1–4), soluble (lanes 5–8), and membrane (lanes 9–12) fractions were separated by SDS/PAGE and immunoblotted with anti-Myc antibodies.