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A role for the DnaJ homologue Scj1p in protein folding in the yeast endoplasmic reticulum.

Silberstein S, Schlenstedt G, Silver PA, Gilmore R - J. Cell Biol. (1998)

Bottom Line: Here, we show that the Deltascj1 mutant is hypersensitive to tunicamycin or mutations that reduce N-linked glycosylation of proteins.Although maturation of glycosylated carboxypeptidase Y occurs with wild-type kinetics in Deltascj1 cells, the transport rate for an unglycosylated mutant carboxypeptidase Y (CPY) is markedly reduced.Loss of Scj1p induces the unfolded protein response pathway, and results in a cell wall defect when combined with an oligosaccharyltransferase mutation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0103, USA.

ABSTRACT
Members of the eukaryotic heat shock protein 70 family (Hsp70s) are regulated by protein cofactors that contain domains homologous to bacterial DnaJ. Of the three DnaJ homologues in the yeast rough endoplasmic reticulum (RER; Scj1p, Sec63p, and Jem1p), Scj1p is most closely related to DnaJ, hence it is a probable cofactor for Kar2p, the major Hsp70 in the yeast RER. However, the physiological role of Scj1p has remained obscure due to the lack of an obvious defect in Kar2p-mediated pathways in scj1 mutants. Here, we show that the Deltascj1 mutant is hypersensitive to tunicamycin or mutations that reduce N-linked glycosylation of proteins. Although maturation of glycosylated carboxypeptidase Y occurs with wild-type kinetics in Deltascj1 cells, the transport rate for an unglycosylated mutant carboxypeptidase Y (CPY) is markedly reduced. Loss of Scj1p induces the unfolded protein response pathway, and results in a cell wall defect when combined with an oligosaccharyltransferase mutation. The combined loss of both Scj1p and Jem1p exaggerates the sensitivity to hypoglycosylation stress, leads to further induction of the unfolded protein response pathway, and drastically delays maturation of an unglycosylated reporter protein in the RER. We propose that the major role for Scj1p is to cooperate with Kar2p to mediate maturation of proteins in the RER lumen.

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Complexes between Kar2p and ER precursor form of unglycosylated CPY. Cultures of the  wild-type (A, RGY141; B,  RGY131), Δscj1 (A, RGY142)  and Δscj1Δjem1 (B, RGY148)  strains bearing pJW373 were  radiolabeled for 10 min at  25°C, and chased for 0 or 60  min. Glass bead extracts of  the labeled cultures were treated with apyrase to deplete ATP. One aliquot from each sample was immunoprecipitated in nondenaturing buffers using antibodies to Kar2p, while a second aliquot was immunoprecipitated with antibodies to CPY. The Kar2p immunoprecipitates were denatured and precipitated with antibodies to CPY. The CPY immunoprecipitates were resolved by PAGE in SDS. The  radioactive bands corresponding to pug-CPY was quantified to determine the percentage of p1CPY and pug-CPY that was associated  with Kar2p. The radioactive band that migrates closely to ug-CPY (e.g., see 0 min of chase) is an artifact caused by compression of the  background by the 50-kD IgG heavy chain.
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Figure 5: Complexes between Kar2p and ER precursor form of unglycosylated CPY. Cultures of the wild-type (A, RGY141; B, RGY131), Δscj1 (A, RGY142) and Δscj1Δjem1 (B, RGY148) strains bearing pJW373 were radiolabeled for 10 min at 25°C, and chased for 0 or 60 min. Glass bead extracts of the labeled cultures were treated with apyrase to deplete ATP. One aliquot from each sample was immunoprecipitated in nondenaturing buffers using antibodies to Kar2p, while a second aliquot was immunoprecipitated with antibodies to CPY. The Kar2p immunoprecipitates were denatured and precipitated with antibodies to CPY. The CPY immunoprecipitates were resolved by PAGE in SDS. The radioactive bands corresponding to pug-CPY was quantified to determine the percentage of p1CPY and pug-CPY that was associated with Kar2p. The radioactive band that migrates closely to ug-CPY (e.g., see 0 min of chase) is an artifact caused by compression of the background by the 50-kD IgG heavy chain.

Mentions: Loss of the Scj1p selectively delays export of unglycosylated CPY from the ER. Presumably, the ability of Kar2p to mediate pug-CPY folding is impaired when one or more of the DnaJ homologues are not expressed. Based upon previous studies using conditional kar2 mutants (te Heesen et al., 1994; Simons et al., 1995), we would predict that the unfolded pug-CPY remains associated with Kar2p for an extended period in Δscj1 and Δscj1Δjem1 cells. To test this prediction, antibodies specific for Kar2p were used to immunoprecipitate the chaperone under nondenaturing conditions together with any unfolded protein substrates. A subsequent immunoprecipitation under denaturing conditions using antibodies to CPY allowed an estimation of the fraction of p1CPY and pug-CPY that were associated with Kar2p either 0 or 60 min after pulse labeling (Fig. 5). Although a faint p1CPY band could be detected in the Kar2p immunoprecipitates from the 0-min chase, quantitation of the data revealed that <5% of the total p1CPY was associated with Kar2p in wild-type, Δscj1 and Δscj1Δjem1 cells. A greater fraction of pug-CPY was associated with Kar2p in wild-type cells immediately after pulse labeling (Fig. 5 A [8%] and B [13%]). After a 1-h chase, neither p1CPY nor pug-CPY remained bound to Kar2p in wild-type cells, nor did we observe artifactual coimmunoprecipitation of p2-CPY or mature CPY with Kar2p in 0 min or 60 min samples. Typically, a slightly greater fraction of the pug-CPY was associated with Kar2p in the Δscj1 (Fig. 5 A, 11%) and Δscj1Δjem1 (Fig. 5 B, 12%) mutants than in the wild-type strain immediately after pulse labeling. In contrast to what we observe in wild-type cells, complexes between Kar2p and pug-CPY persist in the Δscj1 and Δscj1Δjem1 mutants. Quantification of the data in Fig. 5 B indicated that 21% of the remaining pug-CPY was bound to Kar2p in the Δscj1Δjem1 mutant after a 1-h chase. The native coimmunoprecipitation procedure may underestimate the fraction of unfolded proteins that are bound to Kar2p, given that chaperone–substrate complexes may dissociate during the time required for the immunoprecipitation.


A role for the DnaJ homologue Scj1p in protein folding in the yeast endoplasmic reticulum.

Silberstein S, Schlenstedt G, Silver PA, Gilmore R - J. Cell Biol. (1998)

Complexes between Kar2p and ER precursor form of unglycosylated CPY. Cultures of the  wild-type (A, RGY141; B,  RGY131), Δscj1 (A, RGY142)  and Δscj1Δjem1 (B, RGY148)  strains bearing pJW373 were  radiolabeled for 10 min at  25°C, and chased for 0 or 60  min. Glass bead extracts of  the labeled cultures were treated with apyrase to deplete ATP. One aliquot from each sample was immunoprecipitated in nondenaturing buffers using antibodies to Kar2p, while a second aliquot was immunoprecipitated with antibodies to CPY. The Kar2p immunoprecipitates were denatured and precipitated with antibodies to CPY. The CPY immunoprecipitates were resolved by PAGE in SDS. The  radioactive bands corresponding to pug-CPY was quantified to determine the percentage of p1CPY and pug-CPY that was associated  with Kar2p. The radioactive band that migrates closely to ug-CPY (e.g., see 0 min of chase) is an artifact caused by compression of the  background by the 50-kD IgG heavy chain.
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Figure 5: Complexes between Kar2p and ER precursor form of unglycosylated CPY. Cultures of the wild-type (A, RGY141; B, RGY131), Δscj1 (A, RGY142) and Δscj1Δjem1 (B, RGY148) strains bearing pJW373 were radiolabeled for 10 min at 25°C, and chased for 0 or 60 min. Glass bead extracts of the labeled cultures were treated with apyrase to deplete ATP. One aliquot from each sample was immunoprecipitated in nondenaturing buffers using antibodies to Kar2p, while a second aliquot was immunoprecipitated with antibodies to CPY. The Kar2p immunoprecipitates were denatured and precipitated with antibodies to CPY. The CPY immunoprecipitates were resolved by PAGE in SDS. The radioactive bands corresponding to pug-CPY was quantified to determine the percentage of p1CPY and pug-CPY that was associated with Kar2p. The radioactive band that migrates closely to ug-CPY (e.g., see 0 min of chase) is an artifact caused by compression of the background by the 50-kD IgG heavy chain.
Mentions: Loss of the Scj1p selectively delays export of unglycosylated CPY from the ER. Presumably, the ability of Kar2p to mediate pug-CPY folding is impaired when one or more of the DnaJ homologues are not expressed. Based upon previous studies using conditional kar2 mutants (te Heesen et al., 1994; Simons et al., 1995), we would predict that the unfolded pug-CPY remains associated with Kar2p for an extended period in Δscj1 and Δscj1Δjem1 cells. To test this prediction, antibodies specific for Kar2p were used to immunoprecipitate the chaperone under nondenaturing conditions together with any unfolded protein substrates. A subsequent immunoprecipitation under denaturing conditions using antibodies to CPY allowed an estimation of the fraction of p1CPY and pug-CPY that were associated with Kar2p either 0 or 60 min after pulse labeling (Fig. 5). Although a faint p1CPY band could be detected in the Kar2p immunoprecipitates from the 0-min chase, quantitation of the data revealed that <5% of the total p1CPY was associated with Kar2p in wild-type, Δscj1 and Δscj1Δjem1 cells. A greater fraction of pug-CPY was associated with Kar2p in wild-type cells immediately after pulse labeling (Fig. 5 A [8%] and B [13%]). After a 1-h chase, neither p1CPY nor pug-CPY remained bound to Kar2p in wild-type cells, nor did we observe artifactual coimmunoprecipitation of p2-CPY or mature CPY with Kar2p in 0 min or 60 min samples. Typically, a slightly greater fraction of the pug-CPY was associated with Kar2p in the Δscj1 (Fig. 5 A, 11%) and Δscj1Δjem1 (Fig. 5 B, 12%) mutants than in the wild-type strain immediately after pulse labeling. In contrast to what we observe in wild-type cells, complexes between Kar2p and pug-CPY persist in the Δscj1 and Δscj1Δjem1 mutants. Quantification of the data in Fig. 5 B indicated that 21% of the remaining pug-CPY was bound to Kar2p in the Δscj1Δjem1 mutant after a 1-h chase. The native coimmunoprecipitation procedure may underestimate the fraction of unfolded proteins that are bound to Kar2p, given that chaperone–substrate complexes may dissociate during the time required for the immunoprecipitation.

Bottom Line: Here, we show that the Deltascj1 mutant is hypersensitive to tunicamycin or mutations that reduce N-linked glycosylation of proteins.Although maturation of glycosylated carboxypeptidase Y occurs with wild-type kinetics in Deltascj1 cells, the transport rate for an unglycosylated mutant carboxypeptidase Y (CPY) is markedly reduced.Loss of Scj1p induces the unfolded protein response pathway, and results in a cell wall defect when combined with an oligosaccharyltransferase mutation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0103, USA.

ABSTRACT
Members of the eukaryotic heat shock protein 70 family (Hsp70s) are regulated by protein cofactors that contain domains homologous to bacterial DnaJ. Of the three DnaJ homologues in the yeast rough endoplasmic reticulum (RER; Scj1p, Sec63p, and Jem1p), Scj1p is most closely related to DnaJ, hence it is a probable cofactor for Kar2p, the major Hsp70 in the yeast RER. However, the physiological role of Scj1p has remained obscure due to the lack of an obvious defect in Kar2p-mediated pathways in scj1 mutants. Here, we show that the Deltascj1 mutant is hypersensitive to tunicamycin or mutations that reduce N-linked glycosylation of proteins. Although maturation of glycosylated carboxypeptidase Y occurs with wild-type kinetics in Deltascj1 cells, the transport rate for an unglycosylated mutant carboxypeptidase Y (CPY) is markedly reduced. Loss of Scj1p induces the unfolded protein response pathway, and results in a cell wall defect when combined with an oligosaccharyltransferase mutation. The combined loss of both Scj1p and Jem1p exaggerates the sensitivity to hypoglycosylation stress, leads to further induction of the unfolded protein response pathway, and drastically delays maturation of an unglycosylated reporter protein in the RER. We propose that the major role for Scj1p is to cooperate with Kar2p to mediate maturation of proteins in the RER lumen.

Show MeSH