ERAD defects correlate with aggregation of ΔGpαF. (A) Radiolabeled ΔGppαF was translocated into microsomes prepared from wild-type (a), kar2-159 (b), Δjem1 Δscj1 (c), or sec63-1 (d) strains. After translocation, membranes were incubated on ice (○) or at 37°C (•) for 20 min in the presence of ATP. Cytosol prepared from the wild-type strain was added to a final concentration of 5 mg protein/ml, and posttranslocation chase reactions were performed at 37°C for the indicated times. Proteins were precipitated with trichloroacetic acid and analyzed by SDS-PAGE. Relative amounts of ΔGpαF were quantified by radioimaging. Error bars indicate standard deviation from the means of at least four independent experiments. The amount of ΔGpαF at 0 min chase was set to 100%. (B) Radiolabeled ΔGppαF was translocated into microsomes prepared from wild-type (•), kar2-159 (▪), Δjem1 Δscj1 (□), or sec63-1 (○) strains. Membranes were recovered and incubated at 37°C in the presence of ATP for the indicated times. Cytosol was added to a final concentration of 5 mg protein/ml, and posttranslocation chase reactions were performed at 37°C for 20 min. Degradation activity was represented as the amount of ΔGpαF degraded, and the amount of ΔGpαF with 0 min preincubation was set to 100%. (C) Radiolabeled ΔGppαF and ppαF were translocated into microsomes prepared from wild-type (a and e), kar2-159 (b and f), Δjem1 Δscj1 (c and g), or sec63-1 (d and h) strains. After translocation, membranes were incubated at 23°C (a–d) or 37°C (e–h) for 20 min in the presence of ATP. Membranes were solubilized with 1% Triton X-100, subjected to sucrose density gradient centrifugation, and analyzed as in the legend to Fig. 2. Relative amounts of ΔGpαF and glycosylated pαF were quantified by radioimaging. Black bars, ΔGpαF; white bars, glycosylated pαF.

In vivo ERAD analysis of pαF. (A) Wild-type (SEY6210, wt) and Δjem1 Δscj1 (SNY1026-7A, Δjem1 Δscj1) cells were pulse labeled with ³⁵S–amino acids at 23°C for 10 min in the presence of 10 μg/ml tunicamycin, and chased for the indicated times at either 23 or 37°C. pαF was recovered from cell extracts by immunoprecipitation using an anti-ppαF antiserum and analyzed by SDS-PAGE. Relative amounts of pαF were quantified by radioimaging and the means of two independent experiments are shown. The amount of pαF at 0 min chase was set to 100%. □, wild-type chased at 23°C; ○, wild-type chased at 37°C; ▪, Δjem1 Δscj1 chased at 23°C; •, Δjem1 Δscj1 chased at 37°C. (B) kar2-159 (RSY579, kar2-159) and wild-type (SNY1084-1D, wt) cells were pulse labeled with ³⁵S–amino acids for 10 min at 23°C in the presence of 10 μg/ml tunicamycin, and were chased for the times indicated at 23 or 37°C. Degradation of pαF was followed as in A. ▪, kar2-159 chased at 23°C; •, kar2-159 chased at 37°C; □, wild-type chased at 23°C; ○, wild-type chased at 37°C.

Degradation of CPY* in ER chaperone mutants. Wild-type (KYSC1, wt), Δjem1 (KYSC2, Δjem1), Δscj1 (KYSC3, Δscj1), and Δjem1 Δscj1 (KYSC4, Δjem1 Δscj1) cells were pulse labeled with ³⁵S–amino acids for 10 min and chased for the indicated times either at 23°C (A) or at 37°C (B). CPY* was recovered from cell extracts by immunoprecipitation using an anti-CPY antiserum and analyzed by SDS-PAGE. Relative amounts of CPY* were quantified by radioimaging, and the means of two independent experiments are shown. The amount of CPY* at 0 min chase was set to 100%. ▪, wild-type; □, Δjem1; •, Δscj1; ○, Δjem1 Δscj1. (C) kar2-159 (KYSC13, kar2-159) and wild-type (SNY1083, wt) cells were pulse labeled with ³⁵S–amino acids for 10 min at 23°C and chased for the times indicated at 23°C or at 37°C. Degradation of CPY* was followed as in A and B. ▪, kar2-159 chased at 23°C; •, kar2-159 chased at 37°C; □, wild-type chased at 23°C; ○, wild-type chased at 37°C. (D) Δlhs1 (KYSC14) cells and wild-type (KYSC1, wt) cells were pulse labeled with ³⁵S–amino acids for 10 min and chased for the times indicated either at 23 or 37°C. Degradation of CPY* was followed as in A and B. ▪, Δlhs + at 23°C; •, Δlhs1 at 37°C; □, wild-type at 23°C; ○, wild-type at 37°C. (E) sec63-1 (KYSC5, sec63-1) and wild-type (KYSC1, wt) cells were pulse labeled with ³⁵S–amino acids for 10 min at 23°C and chased for the indicated times at 23 or 37°C. Degradation of CPY* was followed as in A and B. ▪, sec63-1 chased at 23°C; •, sec63-1 chased at 37°C; □, wild-type at 23°C; ○, wild-type at 37°C; pp, prepro-CPY*; p1, p1CPY*.

CPY* aggregates in the Δjem1 Δscj1 and the kar2-159 mutants. Wild-type (KYSC1, wt), Δjem1 Δscj1 (KYSC4), and kar2-159 (KYSC13) cells were grown to early logarithmic phase at 23°C and then shifted to 37°C for 60 min. Cell extracts were prepared and subjected to sucrose density gradient centrifugation as described in the Materials and Methods. Fractions were collected and proteins were analyzed by SDS-PAGE followed by immunoblotting using an anti-CPY antiserum. Relative amounts of CPY* were quantified. Numbers indicate fractions (from bottom to top) and P represents the pellet fraction recovered in the bottom of the tube. Vertical arrowheads show the positions of apoferritin (440 kD), alcohol dehydrogenase (150 kD), bovine serum albumin (70 kD), and carbonic anhydrase (30 kD).

Jem1p and Scj1p are dispensable for the degradation of Sec61-2p. (A) Δerg6 cells with the sec61-2 background (SNY1081) were pulse labeled with ³⁵S–amino acids for 10 min, and chased for the times indicated above the lanes at 38°C in the presence (MG132) or the absence (DMSO) of 200 μM MG132. Sec61-2p was recovered from cell extracts by immunoprecipitation using an anti-Sec61p antiserum and analyzed by SDS-PAGE. Relative amounts of Sec61-2p were quantified by radioimaging and the means of two independent experiments are shown. The amount of Sec61-2p at 0 min chase was set to 100%. •, pulse–chase experiment in the absence of MG132; ○, pulse–chase experiments in the presence of 200 μM MG132. (B) Δdoa4 (SNY1082) and its isogenic wild-type (DOA4, RDM15-5B) with the sec61-2 background were pulse labeled with ³⁵S–amino acids for 10 min, chased for the times indicated above the lanes at 38°C, and analyzed as in A. The means of four independent experiments are shown. The amount of Sec61-2p at 0 min chase was set to 100%. •, wild-type; ○, Δdoa4. (C) Wild-type (RDM15-5B), Δjem1 (KYSC11), Δscj1 (SNY1070), and Δjem1 Δscj1 (SNY1071) cells with the sec61-2 background were pulse labeled with ³⁵S–amino acids for 10 min, chased for the times indicated above the lanes at 38°C, and analyzed as in A. The means of two independent experiments are shown. The amount of Sec61-2p at 0 min chase was set to 100%. ▪, wild-type; □, Δjem1; •, Δscj1; ○, Δjem1 Δscj11. (D) ssa1-45 (SNY1078) and its isogenic wild-type (SSA1, SNY1077) strains with the sec61-2 background were pulse labeled with ³⁵S–amino acids for 10 min, chased for the times indicated above the lanes at 37°C, and analyzed as in A. The means of two independent experiments are shown. The amount of Sec61-2p at 0 min chase was set to 100%. •, wild-type; ○, ssa1-45.