Yos9p assists in the degradation of certain nonglycosylated proteins from the endoplasmic reticulum

Abstract

The HRD ubiquitin ligase recognizes and ubiquitylates proteins of the endoplasmic reticulum that display structural defects. Here, we apply quantitative proteomics to characterize the substrate spectrum of the HRD complex. Among the identified substrates is Erg3p, a glycoprotein involved in sterol synthesis. We characterize Erg3p and demonstrate that the elimination of Erg3p requires Htm1p and Yos9p, two proteins that take part in the glycan-dependent turnover of aberrant proteins. We further show that the HRD ligase also mediates the breakdown of Erg3p and CPY* engineered to lack N-glycans. The degradation of these nonglycosylated substrates is enhanced by a mutant variant of Yos9p that has lost its affinity for oligosaccharides, indicating that Yos9p has a previously unrecognized role in the quality control of nonglycosylated proteins.

FIGURE 1:

Membrane topology and posttranslational modifications of Erg3p. (A) A protease protection assay was carried out with extracts from yeast cells expressing either N- or C-terminally tagged Erg3p. The extracts were either left untreated or incubated with 0.3 mg/ml proteinase K. Additionally, detergent was added where indicated. Subsequently, samples were separated by SDS–PAGE and subjected to immunoblotting with the indicated antibodies. Integrity of the vesicles and activity of the protease were controlled by immunoblotting against luminal Kar2p and cytosolically exposed Ubc6p. Protease treatment increased the electrophoretic mobility of HA-Erg3p (A, lane 5). Consequently, the C terminus of Erg3p is protease accessible. (B) Erg3p-myc was immunoprecipitated from solubilized membranes expressing either Erg3p-myc or a variant termed Erg3p-N45Q-myc that carried a mutation in the potential glycosylation site. Precipitated Erg3p was treated with N-glycosidase F where indicated and analyzed by immunoblotting. (C) Predicted membrane topology of Erg3p. Gray boxes illustrate transmembrane segments; black circles indicate the positions of the histidines required for Erg3p function.

FIGURE 2:

Erg3p is short-lived in the presence of Hrd1p. (A) Cells of the indicated strains expressing Erg3p-myc were treated with cycloheximide, and equal aliquots were removed at the specified time points. To analyze Erg3p-myc stability, protein extracts were separated by SDS–PAGE followed by immunoblotting. Sec61p served as loading control. The asterisk (*) indicates background bands that cross-react with the a-Ubc6 antibody. (B) Microsomes were isolated from cells expressing HA-Hrd3p and Erg3p-myc as indicated, and solubilized with NP40 lysis buffer, and HA-Hrd3p was immunoprecipitated. Samples from the total lysates (left panel) and the precipitates (right panel) were separated by SDS–PAGE followed by immunoblotting using the indicated antibodies. (Δ) denotes deletion of the respective gene. (hc) refers to the heavy chain of the a-HA antibody used for precipitation.

FIGURE 3:

Degradation of Erg3p requires Htm1p and Yos9p but not Der1p. (A) Exponentially growing cells of the indicated yeast strains were pulse-labeled with ³⁵S for 15 min, and equal aliquots were removed at the specified time points. Erg3p-myc was immunoprecipitated from cell lysates, and samples were subsequently separated by SDS–PAGE. A PhosphorImager scan of a typical gel is shown. (B) Erg3p-myc degradation was analyzed in the indicated yeast strains. Five independent experiments were quantified using a PhosphorImager, and the results were averaged. The errors bars indicate the SE of the experiments. (C) Stability of CPY* was analyzed in the indicated yeast strains. At least four independent experiments were quantified for each strain using a PhosphorImager, and the results were averaged. The errors bars indicate the SE of the experiments.

FIGURE 4:

Elimination of nonglycosylated substrates by the ER quality control. (A) Pulse-chase experiments were performed to analyze the stability of Erg3p-N45Q-myc in the indicated strain backgrounds. Five independent experiments were quantified using a PhosphorImager, and the results were averaged. For comparison, a degradation curve of glycosylated Erg3p in control cells is included. The errors bars indicate the SE of the experiments. (B) Same as in (A), but CPY*0000 was used as substrate.

FIGURE 5:

Interaction of CPY0000 with the HRD ligase does not prompt its disposal. (A) Microsomes from cells expressing HA-Hrd3p and either CPY*0000 or CPY0000 as indicated were lysed in NP40 buffer, and CPY was precipitated. Samples of the total lysates (lanes 1–3) and the precipitates (lanes 4–6) were separated by SDS–PAGE and tested for the presence of CPY or HA-Hrd3 by immunoblotting. CPY^ER denotes the ER form of CPY, whereas the faster migrating vacuolar form is labeled CPY^Vac. (B) Stability of CPY0000 in control and Δhrd3 cells. Exponentially growing cells of the indicated yeast strains expressing CPY0000 were pulse-labeled with ³⁵S for 10 min, and equal aliquots were removed at the specified time points. CPY was immunoprecipitated from cell lysates. Precipitates were separated by SDS–PAGE, and the gel was scanned using a PhosphorImager.