Export of a cysteine-free misfolded secretory protein from the endoplasmic reticulum for degradation requires interaction with protein disulfide isomerase

Abstract

Protein disulfide isomerase (PDI) interacts with secretory proteins, irrespective of their thiol content, late during translocation into the ER; thus, PDI may be part of the quality control machinery in the ER. We used yeast pdi1 mutants with deletions in the putative peptide binding region of the molecule to investigate its role in the recognition of misfolded secretory proteins in the ER and their export to the cytosol for degradation. Our pdi1 deletion mutants are deficient in the export of a misfolded cysteine-free secretory protein across the ER membrane to the cytosol for degradation, but ER-to-Golgi complex transport of properly folded secretory proteins is only marginally affected. We demonstrate by chemical cross-linking that PDI specifically interacts with the misfolded secretory protein and that mutant forms of PDI have a lower affinity for this protein. In the ER of the pdi1 mutants, a higher proportion of the misfolded secretory protein remains associated with BiP, and in export-deficient sec61 mutants, the misfolded secretory protein remain bounds to PDI. We conclude that the chaperone PDI is part of the quality control machinery in the ER that recognizes terminally misfolded secretory proteins and targets them to the export channel in the ER membrane.

Figure 1

Expression of PDI mutants Δ222–302 and Δ252–277. a, Structures of wild-type S. cerevisiae PDI and deletion mutants Δ222–302 and Δ252–277. b, Mutant PDI proteins are overexpressed. Equal amounts of wild-type and mutant microsomal protein (20 μg/lane) were resolved on a 7.5% SDS gel, and transferred to nitrocellulose. PDI wild-type and mutant proteins, and BiP were detected by incubation with the respective antibodies (1:500 for PDI, 1:1,000 for BiP) ¹²⁵I-labeled protein A, and PhosphorImaging. c, A fraction of wild-type and mutant PDI proteins are underglycosylated. Cells were pulse-labeled with ³⁵S-methionine/cysteine for 15 min at 30°C, lysed, and PDI immunoprecipitated. Immunoprecipitates were incubated in the absence or presence of endoglycosidase H for 4 h at 37°C. Immunoprecipitated proteins were resolved on a 7.5% SDS gel and detected by PhosphorImaging.

Figure 1

Expression of PDI mutants Δ222–302 and Δ252–277. a, Structures of wild-type S. cerevisiae PDI and deletion mutants Δ222–302 and Δ252–277. b, Mutant PDI proteins are overexpressed. Equal amounts of wild-type and mutant microsomal protein (20 μg/lane) were resolved on a 7.5% SDS gel, and transferred to nitrocellulose. PDI wild-type and mutant proteins, and BiP were detected by incubation with the respective antibodies (1:500 for PDI, 1:1,000 for BiP) ¹²⁵I-labeled protein A, and PhosphorImaging. c, A fraction of wild-type and mutant PDI proteins are underglycosylated. Cells were pulse-labeled with ³⁵S-methionine/cysteine for 15 min at 30°C, lysed, and PDI immunoprecipitated. Immunoprecipitates were incubated in the absence or presence of endoglycosidase H for 4 h at 37°C. Immunoprecipitated proteins were resolved on a 7.5% SDS gel and detected by PhosphorImaging.

Figure 1

Expression of PDI mutants Δ222–302 and Δ252–277. a, Structures of wild-type S. cerevisiae PDI and deletion mutants Δ222–302 and Δ252–277. b, Mutant PDI proteins are overexpressed. Equal amounts of wild-type and mutant microsomal protein (20 μg/lane) were resolved on a 7.5% SDS gel, and transferred to nitrocellulose. PDI wild-type and mutant proteins, and BiP were detected by incubation with the respective antibodies (1:500 for PDI, 1:1,000 for BiP) ¹²⁵I-labeled protein A, and PhosphorImaging. c, A fraction of wild-type and mutant PDI proteins are underglycosylated. Cells were pulse-labeled with ³⁵S-methionine/cysteine for 15 min at 30°C, lysed, and PDI immunoprecipitated. Immunoprecipitates were incubated in the absence or presence of endoglycosidase H for 4 h at 37°C. Immunoprecipitated proteins were resolved on a 7.5% SDS gel and detected by PhosphorImaging.

Figure 2

Peptide binding to PDI mutants Δ252–277 and Δ222–302 is reduced, compared with wild-type. ¹²⁵I-labeled glycosylation acceptor peptide, N-benzoyl-NYT-amide, was translocated into PDI wild-type or mutant microsomes as described in Materials and Methods and cross-linked with longwave UV light. All samples were analyzed in duplicate. Proteins were resolved on 7.5% SDS gels and peptide–cross-linking products were visualized by autoradiography. Peptide binding to PDI was quantitated using a PhosphorImager (BioRad).

Figure 3

Effects of deletions in PDI on transport through the secretory pathway. a, Wild-type and mutant cells were pulse-labeled for 10 min at 30°C with ³⁵S-methionine/cysteine, followed by chase incubations for the indicated periods of time. At each time point, cells were lysed and CPY immunoprecipitated. Proteins were resolved on a 7.5% SDS gel and visualized by PhosphorImaging. b, SICs were prepared from wild-type cells and PDI deletion mutants. In vitro translated, radiolabeled ppαf was translocated into the ER of these SICs, and cells were incubated in the presence of ATP, an ATP-regenerating system, and 120 μg/25 μl reaction yeast cytosol for the indicated periods of time at 24°C. Budded vesicles containing gpαf were separated from SICs by differential centrifugation; radiolabeled, glycosylated gpαf was isolated from vesicle and SIC fractions by ConA precipitation, and quantified by scintillation counting.

Figure 3

Effects of deletions in PDI on transport through the secretory pathway. a, Wild-type and mutant cells were pulse-labeled for 10 min at 30°C with ³⁵S-methionine/cysteine, followed by chase incubations for the indicated periods of time. At each time point, cells were lysed and CPY immunoprecipitated. Proteins were resolved on a 7.5% SDS gel and visualized by PhosphorImaging. b, SICs were prepared from wild-type cells and PDI deletion mutants. In vitro translated, radiolabeled ppαf was translocated into the ER of these SICs, and cells were incubated in the presence of ATP, an ATP-regenerating system, and 120 μg/25 μl reaction yeast cytosol for the indicated periods of time at 24°C. Budded vesicles containing gpαf were separated from SICs by differential centrifugation; radiolabeled, glycosylated gpαf was isolated from vesicle and SIC fractions by ConA precipitation, and quantified by scintillation counting.

Figure 4

PDI deletion mutants are defective in export of misfolded secretory proteins from the ER to the cytosol for degradation. a, Export and degradation are dependent on ATP and cytosolic proteasomes. Mutant pΔgpαf was translocated into wild-type microsomes for 50 min at 24°C. Membranes were washed and incubated in the presence or absence of ATP, an ATP-regenerating system, and 6 mg/ml wild-type or proteasome mutant (pre1 pre2) yeast cytosol for the indicated periods of time at 24°C. At each time point, proteins were precipitated with trichloro-acetic acid, and resolved on an 18% polyacrylamide gel containing 4 M urea. Radiolabeled protein was visualized and quantified by PhosphorImaging. A fraction of pΔgpαf (upper band) remains aggregated on the cytosolic face on the microsomes, cannot be removed by buffer washes, and is partially susceptible to proteolysis by cytosolic proteasomes. b, Microsomes were prepared from wild-type cells and pdi1 mutants. Mutant pΔgpαf was translocated into wild-type and mutant microsomes and membranes were incubated in the presence of ATP, an ATP-regenerating system, and 6 mg/ml wild-type yeast cytosol for the indicated periods of time at 24°C. Samples were analyzed as for a. c, The Δgpαf bands from two (wild-type) or four (mutants) degradation experiments performed as in b were quantified using a PhosphorImager (BioRad). Variation at each time point was <10% for wild-type and <5% for the mutants. d, PDI1 wild-type and mutant cells expressing prc1-1 were pulse-labeled for 10 min, chased, and CPY* immunoprecipitated and analyzed as described for CPY in Fig. 3 a. Note that in Δ222–302 and 252–277, an increased proportion of CPY* is transported to the Golgi complex (p2CPY*). p2CPY* is not secreted, and is rapidly degraded in PEP4 wild-type cells, but relatively stable in pep4::URA3 cells (shown here).

Figure 4

PDI deletion mutants are defective in export of misfolded secretory proteins from the ER to the cytosol for degradation. a, Export and degradation are dependent on ATP and cytosolic proteasomes. Mutant pΔgpαf was translocated into wild-type microsomes for 50 min at 24°C. Membranes were washed and incubated in the presence or absence of ATP, an ATP-regenerating system, and 6 mg/ml wild-type or proteasome mutant (pre1 pre2) yeast cytosol for the indicated periods of time at 24°C. At each time point, proteins were precipitated with trichloro-acetic acid, and resolved on an 18% polyacrylamide gel containing 4 M urea. Radiolabeled protein was visualized and quantified by PhosphorImaging. A fraction of pΔgpαf (upper band) remains aggregated on the cytosolic face on the microsomes, cannot be removed by buffer washes, and is partially susceptible to proteolysis by cytosolic proteasomes. b, Microsomes were prepared from wild-type cells and pdi1 mutants. Mutant pΔgpαf was translocated into wild-type and mutant microsomes and membranes were incubated in the presence of ATP, an ATP-regenerating system, and 6 mg/ml wild-type yeast cytosol for the indicated periods of time at 24°C. Samples were analyzed as for a. c, The Δgpαf bands from two (wild-type) or four (mutants) degradation experiments performed as in b were quantified using a PhosphorImager (BioRad). Variation at each time point was <10% for wild-type and <5% for the mutants. d, PDI1 wild-type and mutant cells expressing prc1-1 were pulse-labeled for 10 min, chased, and CPY* immunoprecipitated and analyzed as described for CPY in Fig. 3 a. Note that in Δ222–302 and 252–277, an increased proportion of CPY* is transported to the Golgi complex (p2CPY*). p2CPY* is not secreted, and is rapidly degraded in PEP4 wild-type cells, but relatively stable in pep4::URA3 cells (shown here).

Figure 4

PDI deletion mutants are defective in export of misfolded secretory proteins from the ER to the cytosol for degradation. a, Export and degradation are dependent on ATP and cytosolic proteasomes. Mutant pΔgpαf was translocated into wild-type microsomes for 50 min at 24°C. Membranes were washed and incubated in the presence or absence of ATP, an ATP-regenerating system, and 6 mg/ml wild-type or proteasome mutant (pre1 pre2) yeast cytosol for the indicated periods of time at 24°C. At each time point, proteins were precipitated with trichloro-acetic acid, and resolved on an 18% polyacrylamide gel containing 4 M urea. Radiolabeled protein was visualized and quantified by PhosphorImaging. A fraction of pΔgpαf (upper band) remains aggregated on the cytosolic face on the microsomes, cannot be removed by buffer washes, and is partially susceptible to proteolysis by cytosolic proteasomes. b, Microsomes were prepared from wild-type cells and pdi1 mutants. Mutant pΔgpαf was translocated into wild-type and mutant microsomes and membranes were incubated in the presence of ATP, an ATP-regenerating system, and 6 mg/ml wild-type yeast cytosol for the indicated periods of time at 24°C. Samples were analyzed as for a. c, The Δgpαf bands from two (wild-type) or four (mutants) degradation experiments performed as in b were quantified using a PhosphorImager (BioRad). Variation at each time point was <10% for wild-type and <5% for the mutants. d, PDI1 wild-type and mutant cells expressing prc1-1 were pulse-labeled for 10 min, chased, and CPY* immunoprecipitated and analyzed as described for CPY in Fig. 3 a. Note that in Δ222–302 and 252–277, an increased proportion of CPY* is transported to the Golgi complex (p2CPY*). p2CPY* is not secreted, and is rapidly degraded in PEP4 wild-type cells, but relatively stable in pep4::URA3 cells (shown here).

Figure 4

PDI deletion mutants are defective in export of misfolded secretory proteins from the ER to the cytosol for degradation. a, Export and degradation are dependent on ATP and cytosolic proteasomes. Mutant pΔgpαf was translocated into wild-type microsomes for 50 min at 24°C. Membranes were washed and incubated in the presence or absence of ATP, an ATP-regenerating system, and 6 mg/ml wild-type or proteasome mutant (pre1 pre2) yeast cytosol for the indicated periods of time at 24°C. At each time point, proteins were precipitated with trichloro-acetic acid, and resolved on an 18% polyacrylamide gel containing 4 M urea. Radiolabeled protein was visualized and quantified by PhosphorImaging. A fraction of pΔgpαf (upper band) remains aggregated on the cytosolic face on the microsomes, cannot be removed by buffer washes, and is partially susceptible to proteolysis by cytosolic proteasomes. b, Microsomes were prepared from wild-type cells and pdi1 mutants. Mutant pΔgpαf was translocated into wild-type and mutant microsomes and membranes were incubated in the presence of ATP, an ATP-regenerating system, and 6 mg/ml wild-type yeast cytosol for the indicated periods of time at 24°C. Samples were analyzed as for a. c, The Δgpαf bands from two (wild-type) or four (mutants) degradation experiments performed as in b were quantified using a PhosphorImager (BioRad). Variation at each time point was <10% for wild-type and <5% for the mutants. d, PDI1 wild-type and mutant cells expressing prc1-1 were pulse-labeled for 10 min, chased, and CPY* immunoprecipitated and analyzed as described for CPY in Fig. 3 a. Note that in Δ222–302 and 252–277, an increased proportion of CPY* is transported to the Golgi complex (p2CPY*). p2CPY* is not secreted, and is rapidly degraded in PEP4 wild-type cells, but relatively stable in pep4::URA3 cells (shown here).

Figure 4

PDI deletion mutants are defective in export of misfolded secretory proteins from the ER to the cytosol for degradation. a, Export and degradation are dependent on ATP and cytosolic proteasomes. Mutant pΔgpαf was translocated into wild-type microsomes for 50 min at 24°C. Membranes were washed and incubated in the presence or absence of ATP, an ATP-regenerating system, and 6 mg/ml wild-type or proteasome mutant (pre1 pre2) yeast cytosol for the indicated periods of time at 24°C. At each time point, proteins were precipitated with trichloro-acetic acid, and resolved on an 18% polyacrylamide gel containing 4 M urea. Radiolabeled protein was visualized and quantified by PhosphorImaging. A fraction of pΔgpαf (upper band) remains aggregated on the cytosolic face on the microsomes, cannot be removed by buffer washes, and is partially susceptible to proteolysis by cytosolic proteasomes. b, Microsomes were prepared from wild-type cells and pdi1 mutants. Mutant pΔgpαf was translocated into wild-type and mutant microsomes and membranes were incubated in the presence of ATP, an ATP-regenerating system, and 6 mg/ml wild-type yeast cytosol for the indicated periods of time at 24°C. Samples were analyzed as for a. c, The Δgpαf bands from two (wild-type) or four (mutants) degradation experiments performed as in b were quantified using a PhosphorImager (BioRad). Variation at each time point was <10% for wild-type and <5% for the mutants. d, PDI1 wild-type and mutant cells expressing prc1-1 were pulse-labeled for 10 min, chased, and CPY* immunoprecipitated and analyzed as described for CPY in Fig. 3 a. Note that in Δ222–302 and 252–277, an increased proportion of CPY* is transported to the Golgi complex (p2CPY*). p2CPY* is not secreted, and is rapidly degraded in PEP4 wild-type cells, but relatively stable in pep4::URA3 cells (shown here).

Figure 5

PDI specifically interacts with a misfolded, cysteine-free secretory protein. a, Wild-type PDI preferentially binds to Δgpαf. Radiolabeled pΔgpaf (lanes 1–3) or ppαf (lanes 4 and 5) were translocated into wild-type microsomes. Directly after termination of the translocation reaction, DSP cross-linking was performed as described in Materials and Methods, followed by solubilization of the membranes, and precipitation of glycoproteins with ConA-Sepharose or immunoprecipitation with anti-PDI antiserum. Cross-linked proteins were resolved on nonreducing 7.5% SDS gels and visualized by PhosphorImaging. The positions of major cross-linking products are indicated (PDIxΔgpαf and PDIxgpαf). Note that wild-type gpαf is a glycoprotein, thus, both monomeric gpαf (•, lanes 4 and 5) and gpαf dimers (*, lane 5) bind to ConA-Sepharose, whereas Δgpαf contains no oligosaccharyl side chains and, therefore, only binds to ConA-Sepharose if cross-linked to the glycoprotein PDI (lane 2). b, Radiolabeled pΔgpαf was translocated into wild-type and pdi1 mutant microsomes and cross-linked with DSP as described. Cross-linking products of Δgpαf and wild-type or mutant PDI proteins were detected by precipitation with ConA-Sepharose.

Figure 5

PDI specifically interacts with a misfolded, cysteine-free secretory protein. a, Wild-type PDI preferentially binds to Δgpαf. Radiolabeled pΔgpaf (lanes 1–3) or ppαf (lanes 4 and 5) were translocated into wild-type microsomes. Directly after termination of the translocation reaction, DSP cross-linking was performed as described in Materials and Methods, followed by solubilization of the membranes, and precipitation of glycoproteins with ConA-Sepharose or immunoprecipitation with anti-PDI antiserum. Cross-linked proteins were resolved on nonreducing 7.5% SDS gels and visualized by PhosphorImaging. The positions of major cross-linking products are indicated (PDIxΔgpαf and PDIxgpαf). Note that wild-type gpαf is a glycoprotein, thus, both monomeric gpαf (•, lanes 4 and 5) and gpαf dimers (*, lane 5) bind to ConA-Sepharose, whereas Δgpαf contains no oligosaccharyl side chains and, therefore, only binds to ConA-Sepharose if cross-linked to the glycoprotein PDI (lane 2). b, Radiolabeled pΔgpαf was translocated into wild-type and pdi1 mutant microsomes and cross-linked with DSP as described. Cross-linking products of Δgpαf and wild-type or mutant PDI proteins were detected by precipitation with ConA-Sepharose.

Figure 6

Deletions in PDI result in increased association of misfolded secretory proteins with BiP. a, BiP binds to monomeric and aggregated misfolded secretory proteins. Left, Radiolabeled pΔgpαf (lanes 1 and 2) or ppαf (lanes 3 and 4) were translocated into wild-type microsomes, followed by DSP cross-linking, solubilization, and immunoprecipitation with anti-BiP antibodies. BiP–cross-linked proteins were resolved on nonreducing 7.5% SDS gels and visualized by PhosphorImaging. Note that a fraction of monomeric and dimeric gpαf (•, lanes 3 and 4; *, lane 4) and dimeric Δgpaf (••, lane 2) precipitated nonspecifically with protein A–Sepharose. Right, To reveal the individual constituents of the BiPxΔgpαf cross-linking product, we performed sequential immunoprecipitations of the cross-linked material first with anti-ppαf, then with anti-BiP antiserum. Cross-links were cleaved by incubation in sample buffer containing 100 mM DTT, proteins were resolved on a 7.5% SDS gel, and visualized by silverstaining (lanes 1–3) and autoradiography (lanes 4–6). b, Deletions in PDI result in increased association of misfolded secretory proteins with BiP. Radiolabeled pΔgpαf was translocated into wild-type or pdi1 mutant microsomes as indicated, followed by DSP cross-linking, solubilization, and immunoprecipitation with anti-BiP antibodies (lanes 1–3), or incubation with protein A-Sepharose only (lanes 4–6). To facilitate quantitation of BiP-associated Δgpαf, cross-links were cleaved with DTT before SDS-PAGE on 18% 4 M urea gels.

Figure 6

Deletions in PDI result in increased association of misfolded secretory proteins with BiP. a, BiP binds to monomeric and aggregated misfolded secretory proteins. Left, Radiolabeled pΔgpαf (lanes 1 and 2) or ppαf (lanes 3 and 4) were translocated into wild-type microsomes, followed by DSP cross-linking, solubilization, and immunoprecipitation with anti-BiP antibodies. BiP–cross-linked proteins were resolved on nonreducing 7.5% SDS gels and visualized by PhosphorImaging. Note that a fraction of monomeric and dimeric gpαf (•, lanes 3 and 4; *, lane 4) and dimeric Δgpaf (••, lane 2) precipitated nonspecifically with protein A–Sepharose. Right, To reveal the individual constituents of the BiPxΔgpαf cross-linking product, we performed sequential immunoprecipitations of the cross-linked material first with anti-ppαf, then with anti-BiP antiserum. Cross-links were cleaved by incubation in sample buffer containing 100 mM DTT, proteins were resolved on a 7.5% SDS gel, and visualized by silverstaining (lanes 1–3) and autoradiography (lanes 4–6). b, Deletions in PDI result in increased association of misfolded secretory proteins with BiP. Radiolabeled pΔgpαf was translocated into wild-type or pdi1 mutant microsomes as indicated, followed by DSP cross-linking, solubilization, and immunoprecipitation with anti-BiP antibodies (lanes 1–3), or incubation with protein A-Sepharose only (lanes 4–6). To facilitate quantitation of BiP-associated Δgpαf, cross-links were cleaved with DTT before SDS-PAGE on 18% 4 M urea gels.

Figure 6

Deletions in PDI result in increased association of misfolded secretory proteins with BiP. a, BiP binds to monomeric and aggregated misfolded secretory proteins. Left, Radiolabeled pΔgpαf (lanes 1 and 2) or ppαf (lanes 3 and 4) were translocated into wild-type microsomes, followed by DSP cross-linking, solubilization, and immunoprecipitation with anti-BiP antibodies. BiP–cross-linked proteins were resolved on nonreducing 7.5% SDS gels and visualized by PhosphorImaging. Note that a fraction of monomeric and dimeric gpαf (•, lanes 3 and 4; *, lane 4) and dimeric Δgpaf (••, lane 2) precipitated nonspecifically with protein A–Sepharose. Right, To reveal the individual constituents of the BiPxΔgpαf cross-linking product, we performed sequential immunoprecipitations of the cross-linked material first with anti-ppαf, then with anti-BiP antiserum. Cross-links were cleaved by incubation in sample buffer containing 100 mM DTT, proteins were resolved on a 7.5% SDS gel, and visualized by silverstaining (lanes 1–3) and autoradiography (lanes 4–6). b, Deletions in PDI result in increased association of misfolded secretory proteins with BiP. Radiolabeled pΔgpαf was translocated into wild-type or pdi1 mutant microsomes as indicated, followed by DSP cross-linking, solubilization, and immunoprecipitation with anti-BiP antibodies (lanes 1–3), or incubation with protein A-Sepharose only (lanes 4–6). To facilitate quantitation of BiP-associated Δgpαf, cross-links were cleaved with DTT before SDS-PAGE on 18% 4 M urea gels.

Figure 7

PDI and BiP are associated in the ER lumen and dissociate in the presence of a folding substrate. a, Wild-type and pdi1 mutant microsomes were incubated in translocation reactions containing pΔgpαf (lanes 1–3) or no translocation substrate (lanes 4–6). At the end of the translocation, DSP cross-linking was performed as described above, followed by solubilization of the membranes, and immunoprecipitation with anti-PDI antiserum. Cross-links in the immunoprecipitate were cleaved with DTT, proteins were resolved on 7.5% SDS gels, and transferred to nitrocellulose membranes. BiP was detected by immunoblotting with anti-BiP antiserum (1:1,000) followed by incubation with ¹²⁵I-protein A and PhosphorImaging. b, Quantitation of the experiment shown in a.

Figure 7

PDI and BiP are associated in the ER lumen and dissociate in the presence of a folding substrate. a, Wild-type and pdi1 mutant microsomes were incubated in translocation reactions containing pΔgpαf (lanes 1–3) or no translocation substrate (lanes 4–6). At the end of the translocation, DSP cross-linking was performed as described above, followed by solubilization of the membranes, and immunoprecipitation with anti-PDI antiserum. Cross-links in the immunoprecipitate were cleaved with DTT, proteins were resolved on 7.5% SDS gels, and transferred to nitrocellulose membranes. BiP was detected by immunoblotting with anti-BiP antiserum (1:1,000) followed by incubation with ¹²⁵I-protein A and PhosphorImaging. b, Quantitation of the experiment shown in a.

Figure 8

In export-deficient sec61 mutant microsomes, misfolded secretory proteins remain associated with PDI. Radiolabeled pΔgpαf was translocated into SEC61 wild-type and mutant microsomes as described in Materials and Methods and cross-linked with DSP. PDI– and BiP–cross-linked proteins were immunoprecipitated with the respective antibodies. Cross-links were cleaved with DTT, proteins resolved on 18% polyacrylamide gels containing 4 M urea, and radiolabeled Δgpαf associated with PDI or BiP was visualized by autoradiography and quantified using a PhosphorImager. Relative amounts of Δgpαf associated with PDI and BiP are indicated with and without adjustment for increased levels of BiP and PDI due to the unfolded protein response induction in the sec61 mutant microsomes (1.7× for sec61-32; 1.4× for sec61-41).

Figure 9

Predicted structure of PDI deletion mutants. a, Domain structure of mature yeast PDI and relative positions of deletions. Each rectangle in PDI corresponds to a thioredoxin fold. Domain boundaries are indicated and were determined as described in Materials and Methods. The domain boundaries for b and b′ include the loops linking them to domains a and a′, respectively. Regions shaded in gray correspond to deleted amino acids in the b and b′ domains. b, Predicted structure of Δ222–302. The deleted amino acids were mapped into two thioredoxin structures using the b and b′ domain definitions and assuming the b′ domain has a thioredoxin fold. The two thioredoxin structures were docked manually, as described in Materials and Methods. Deleted residues are in red and remaining residues are in green. The figure shown on the bottom is identical to the one shown on top, but tilted 90° towards the viewer. c, Predicted structure of Δ252–277. Deleted residues (red) were mapped into the thioredoxin structure using the b′ domain boundary definitions described in the text. These figures were obtained using the QUANTA package (molecular modeling software package, 1992).

Figure 9

Predicted structure of PDI deletion mutants. a, Domain structure of mature yeast PDI and relative positions of deletions. Each rectangle in PDI corresponds to a thioredoxin fold. Domain boundaries are indicated and were determined as described in Materials and Methods. The domain boundaries for b and b′ include the loops linking them to domains a and a′, respectively. Regions shaded in gray correspond to deleted amino acids in the b and b′ domains. b, Predicted structure of Δ222–302. The deleted amino acids were mapped into two thioredoxin structures using the b and b′ domain definitions and assuming the b′ domain has a thioredoxin fold. The two thioredoxin structures were docked manually, as described in Materials and Methods. Deleted residues are in red and remaining residues are in green. The figure shown on the bottom is identical to the one shown on top, but tilted 90° towards the viewer. c, Predicted structure of Δ252–277. Deleted residues (red) were mapped into the thioredoxin structure using the b′ domain boundary definitions described in the text. These figures were obtained using the QUANTA package (molecular modeling software package, 1992).

Figure 9

Predicted structure of PDI deletion mutants. a, Domain structure of mature yeast PDI and relative positions of deletions. Each rectangle in PDI corresponds to a thioredoxin fold. Domain boundaries are indicated and were determined as described in Materials and Methods. The domain boundaries for b and b′ include the loops linking them to domains a and a′, respectively. Regions shaded in gray correspond to deleted amino acids in the b and b′ domains. b, Predicted structure of Δ222–302. The deleted amino acids were mapped into two thioredoxin structures using the b and b′ domain definitions and assuming the b′ domain has a thioredoxin fold. The two thioredoxin structures were docked manually, as described in Materials and Methods. Deleted residues are in red and remaining residues are in green. The figure shown on the bottom is identical to the one shown on top, but tilted 90° towards the viewer. c, Predicted structure of Δ252–277. Deleted residues (red) were mapped into the thioredoxin structure using the b′ domain boundary definitions described in the text. These figures were obtained using the QUANTA package (molecular modeling software package, 1992).

Figure 10

Postulated sequence of events after secretory protein translocation into the ER for wild-type and misfolded proteins (see Discussion).