R217A BiP exhibits J domain specificity. A, residue R217 was mapped onto the crystal structure of the ATPase domain of bovine Hsc70 (44) after aligning the protein sequences of BiP and Hsc70. This residue is conserved. B, the ATPase activity of wild-type (●) and R217A BiP (○) at a final concentration of 0.7 μm was measured in the presence of increasing molar ratios of peptide p5. C, the ATPase activity of wild-type or R217A BiP at a final concentration of 0.7 μm was measured in the absence or presence of increasing amounts of the J domains from Sec63p or Jem1p. Data represent the means of a minimum of three independent experiments ± S.E. (error bars). D, the Sec63p complex, which contains BiP, Sec63p, Sec71p, and Sec72p, was purified from microsomes derived from TEF1-KAR2 or TEF1-R217A yeast, resolved by SDS-PAGE, and subjected to Coomassie Brilliant Blue staining or immunoblot analysis. The ratio of BiP to Sec63p from each analysis is indicated below the corresponding set of panels.

Yeast expressing R217A BiP exhibit a defect in protein translocation. A, the ER translocation of ΔGppαF and pre-BiP was analyzed at the indicated time points during a pulse-chase immunoprecipitation experiment using TEF1-KAR2 and TEF1-R217A yeast. The signal peptidase-deficient strain, sec11-7, served as a positive control. B, translocation assays with wild-type ppαF were performed using microsomes derived from TEF1-KAR2 or TEF1-R217A yeast. As controls, reactions either lacked microsomes (−) or contained microsomes derived from the kar2–159 mutant strain. After 60 min, each reaction was aliquoted and treated with buffer (A), trypsin (B), or trypsin and Triton X-100 (C). The percentage of translocation efficiency is indicated below each panel and corresponds to the means from a minimum of four independent experiments. C, the degradation of a soluble ERAD substrate, CPY*, was measured by pulse-chase analysis in the TEF1-KAR2 (●) and TEF1-R217A (○) strains. At each time point, data were standardized to the amount of protein at the start of the chase and represent the means of a minimum of three independent experiments ± S.E. D, ERAD assays were performed to assess the degradation of pαF using microsomes from TEF1-KAR2 or TEF1-R217A yeast. Reactions were performed in the absence (white bars) or presence (gray bars) of an ATP regeneration system and 0.5 mg/ml yeast cytosol. Data were standardized to the amount of protein remaining in the absence of cytosol and ATP regeneration system and represent the means of a minimum of four independent experiments ± S.E. (error bars).

The genetic interactions exhibited by kar2-R217A resemble those exhibited by the sec71Δ and sec72Δ translocation-deficient strains. Genetic interactions were quantified by using a π-score, which represents the difference between the observed double mutant UPR reporter level and that expected in the absence of a genetic interaction (45). A, correlations between the patterns of π-scores of the kar2-R217A, kar2-DAmP, or KAR2 strains and the π-scores of every other mutant in the data set of Jonikas et al. (45) obtained in the UPR-based genetic analysis are depicted using histograms. Select correlations, including those to sec71Δ, sec72Δ, and known components of the ERAD pathway are highlighted. B, a “heat map” comparing select genetic interactions exhibited by the wild-type and mutant kar2 strains with known translocation and ERAD mutants.

The K584X and S493F BiP mutants are defective for peptide-stimulated ATPase activity. A, the Lys⁵⁸⁴ and Ser⁴⁹³ residues were mapped onto the crystal structure of the SBD and lid domain of the bacterial Hsp70 DnaK (68) after aligning the BiP and DnaK protein sequences. Although Lys⁵⁸⁴ is conserved between BiP and DnaK, Ser⁴⁹³ is Ala⁴⁴⁸ in DnaK. B, ATPase assays were performed in the presence of increasing molar ratios of peptide p5 (CLLLSAPRR) (69). Reactions contained wild-type (WT) (●), K584X (▵), or S493F (▿) BiP at a final concentration of 0.7 μm. Data represent the means of a minimum of three independent experiments ± S.E. C, ATPase assays were performed in the absence (white bars) or presence (gray bars) of the J domains from Sec63p (left panel) or Jem1p (right panel), as described (30, 32). The wild-type or mutant BiP proteins (2.1 μm) and the J domains were present in equimolar amounts. Data represent the means of a minimum of three independent experiments ± S.E. (error bars).

Characterization of yeast strains expressing wild-type, K584X, S493F, or R217A BiP from the P_TEF1 promoter. A, 10-fold serial dilutions of yeast expressing wild-type or mutant BiP were plated onto selective medium. The plates were incubated at 26, 30, or 37 °C for 2 days. Where indicated, growth was also tested in the presence of 8 mm DTT. B, to measure the stability of the wild-type and mutant BiP proteins, pulse-chase immunoprecipitation assays were performed with the TEF1-KAR2 (●), TEF1-R217A (○), TEF1-K584X (▵), and TEF1-S493F (▿) strains grown at 30 °C. At each time point, data were standardized to the amount of protein at the start of the chase and represent the means of three or more independent experiments ± S.E. C, UPR induction in the wild-type and indicated mutant strains was analyzed using a β-galactosidase reporter assay. Cells were incubated either at 30 °C (white bars), shifted to 37 °C for 1 h (gray bars), or treated with 8 mm DTT for 1 h at 30 °C (black bars). Data represent the means of two independent experiments ± range of the observed values. The DTT-stimulated UPR induction in the TEF1-KAR2 strain was 2-fold higher than the control, whereas in the TEF1-R217A strain it was 4.5-fold higher than the corresponding control.

Yeast expressing K584X and S493F BiP are translocation- and ERAD-defective. A, the translocation of wild-type ppαF was assessed at 30 °C using microsomes derived from TEF1-KAR2, TEF1-K584X, or TEF1-S493F yeast. After 60 min, each reaction was split and treated either with buffer (A), trypsin (B), or trypsin and Triton X-100 (C). The percentage of translocation efficiency is indicated below each panel. The graph on the right compares the percentage of translocation efficiency of the microsomes when the assay was performed at 20 °C (white bars) or 30 °C (gray bars). Data represent the means of a minimum of four independent experiments ± S.E. (error bars). B, the degradation of CPY*, a soluble ERAD substrate, was measured at 30 °C in the TEF1-KAR2 (●), TEF1-K584X (▵), and TEF1-S493F (▿) strains by pulse-chase analysis. At each time point, data were standardized to the amount of protein at the start of the chase and represent the means of a minimum of three independent experiments ± S.E. C, ERAD assays were performed at 30 °C to assess the degradation of pαF using microsomes from TEF1-KAR2, TEF1-K584X, or TEF1-S493F yeast. Reactions were carried out in the absence (white bars) or presence (gray bars) of an ATP regeneration system and 0.5 mg/ml yeast cytosol. Data were standardized to the amount of protein remaining in the absence of cytosol and the ATP regeneration system and represent the means of a minimum of three independent experiments ± S.E.