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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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Intracellular transport of CPY and unglycosylated CPY  after removal of DTT. Wild-type (RGY141), Δscj1 (RGY142)  and Δscj1Δjem1 (RGY148) strains that were incubated for 15 min  at 25°C in the presence of 5 mM DTT, were pulse labeled for 10  min with Tran-35S-label. The cultures were then diluted 10-fold  with DTT-free medium containing an excess of cold methionine  and cysteine, and aliquots were taken at the indicated times. CPY  was immunoprecipitated from glass bead extracts of the cells and  resolved by PAGE in SDS. The ER, Golgi, and vacuolar forms of  CPY, expressed from the genomic PRC1 gene, are designated as  p1CPY, p2CPY, and CPY, respectively. 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. The precursor and vacuolar forms of unglycosylated CPY encoded by pJW373 are designated as pug-CPY and  ug-CPY. (B) The radiolabeled bands from the gel in A were  quantified, normalized to the total amount of CPY (precursors  plus mature) present in a sample, and expressed as the percentage of mature CPY (left) or mature ug-CPY (right) for each time  point of the chase.
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Figure 8: Intracellular transport of CPY and unglycosylated CPY after removal of DTT. Wild-type (RGY141), Δscj1 (RGY142) and Δscj1Δjem1 (RGY148) strains that were incubated for 15 min at 25°C in the presence of 5 mM DTT, were pulse labeled for 10 min with Tran-35S-label. The cultures were then diluted 10-fold with DTT-free medium containing an excess of cold methionine and cysteine, and aliquots were taken at the indicated times. CPY was immunoprecipitated from glass bead extracts of the cells and resolved by PAGE in SDS. The ER, Golgi, and vacuolar forms of CPY, expressed from the genomic PRC1 gene, are designated as p1CPY, p2CPY, and CPY, respectively. 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. The precursor and vacuolar forms of unglycosylated CPY encoded by pJW373 are designated as pug-CPY and ug-CPY. (B) The radiolabeled bands from the gel in A were quantified, normalized to the total amount of CPY (precursors plus mature) present in a sample, and expressed as the percentage of mature CPY (left) or mature ug-CPY (right) for each time point of the chase.

Mentions: To determine whether Scj1p and Jem1p were also required for the conformational maturation of DTT-reduced p1CPY and pug-CPY, we compared transport of CPY and ug-CPY to the vacuole in wild-type, Δscj1 and Δscj1Δjem1 mutant cells that were radiolabeled in the presence of DTT. In agreement with previous reports (Jämsä et al., 1994; Simons et al., 1995), only the reduced p1 precursor forms of CPY or pug-CPY accumulate when cells are pulse labeled for 30 min in the presence of DTT (Fig. 8 A). After dilution of the reducing agent, p1CPY and pug-CPY are oxidized and fold into transport competent forms, as judged by the disappearance of p1CPY and pug-CPY. Transport of fully glycosylated CPY occurred with very similar kinetics in wild-type and Δscj1 mutant cells (Fig. 8 B, left). We conclude that Scj1p is not required for a rate-limiting step in the folding of DTT-reduced CPY in the endoplasmic reticulum. Exit of p1CPY from the ER was delayed, but not prevented, in theΔscj1Δjem1 mutant relative to the wild-type strain (Fig. 8 B, right). After dilution of the reducing agent, transport of unglycosylated CPY to the vacuole was delayed roughly twofold in the Δscj1 mutant relative to the wild-type strain, just as we had observed in the absence of DTT treatment. The loss of both DnaJ homologues had a much more profound effect upon folding and ER-exit of pug-CPY. We did not detect conversion of pug-CPY to ug-CPY during the 90 minute incubation after dilution of the DTT in the Δscj1Δjem1 mutant (Fig. 8 B, right), suggesting that the DTT-reduced unglycosylated CPY cannot be rescued when the folding machinery is compromised by loss of both Scj1p and Jem1p.


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)

Intracellular transport of CPY and unglycosylated CPY  after removal of DTT. Wild-type (RGY141), Δscj1 (RGY142)  and Δscj1Δjem1 (RGY148) strains that were incubated for 15 min  at 25°C in the presence of 5 mM DTT, were pulse labeled for 10  min with Tran-35S-label. The cultures were then diluted 10-fold  with DTT-free medium containing an excess of cold methionine  and cysteine, and aliquots were taken at the indicated times. CPY  was immunoprecipitated from glass bead extracts of the cells and  resolved by PAGE in SDS. The ER, Golgi, and vacuolar forms of  CPY, expressed from the genomic PRC1 gene, are designated as  p1CPY, p2CPY, and CPY, respectively. 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. The precursor and vacuolar forms of unglycosylated CPY encoded by pJW373 are designated as pug-CPY and  ug-CPY. (B) The radiolabeled bands from the gel in A were  quantified, normalized to the total amount of CPY (precursors  plus mature) present in a sample, and expressed as the percentage of mature CPY (left) or mature ug-CPY (right) for each time  point of the chase.
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Related In: Results  -  Collection

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Figure 8: Intracellular transport of CPY and unglycosylated CPY after removal of DTT. Wild-type (RGY141), Δscj1 (RGY142) and Δscj1Δjem1 (RGY148) strains that were incubated for 15 min at 25°C in the presence of 5 mM DTT, were pulse labeled for 10 min with Tran-35S-label. The cultures were then diluted 10-fold with DTT-free medium containing an excess of cold methionine and cysteine, and aliquots were taken at the indicated times. CPY was immunoprecipitated from glass bead extracts of the cells and resolved by PAGE in SDS. The ER, Golgi, and vacuolar forms of CPY, expressed from the genomic PRC1 gene, are designated as p1CPY, p2CPY, and CPY, respectively. 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. The precursor and vacuolar forms of unglycosylated CPY encoded by pJW373 are designated as pug-CPY and ug-CPY. (B) The radiolabeled bands from the gel in A were quantified, normalized to the total amount of CPY (precursors plus mature) present in a sample, and expressed as the percentage of mature CPY (left) or mature ug-CPY (right) for each time point of the chase.
Mentions: To determine whether Scj1p and Jem1p were also required for the conformational maturation of DTT-reduced p1CPY and pug-CPY, we compared transport of CPY and ug-CPY to the vacuole in wild-type, Δscj1 and Δscj1Δjem1 mutant cells that were radiolabeled in the presence of DTT. In agreement with previous reports (Jämsä et al., 1994; Simons et al., 1995), only the reduced p1 precursor forms of CPY or pug-CPY accumulate when cells are pulse labeled for 30 min in the presence of DTT (Fig. 8 A). After dilution of the reducing agent, p1CPY and pug-CPY are oxidized and fold into transport competent forms, as judged by the disappearance of p1CPY and pug-CPY. Transport of fully glycosylated CPY occurred with very similar kinetics in wild-type and Δscj1 mutant cells (Fig. 8 B, left). We conclude that Scj1p is not required for a rate-limiting step in the folding of DTT-reduced CPY in the endoplasmic reticulum. Exit of p1CPY from the ER was delayed, but not prevented, in theΔscj1Δjem1 mutant relative to the wild-type strain (Fig. 8 B, right). After dilution of the reducing agent, transport of unglycosylated CPY to the vacuole was delayed roughly twofold in the Δscj1 mutant relative to the wild-type strain, just as we had observed in the absence of DTT treatment. The loss of both DnaJ homologues had a much more profound effect upon folding and ER-exit of pug-CPY. We did not detect conversion of pug-CPY to ug-CPY during the 90 minute incubation after dilution of the DTT in the Δscj1Δjem1 mutant (Fig. 8 B, right), suggesting that the DTT-reduced unglycosylated CPY cannot be rescued when the folding machinery is compromised by loss of both Scj1p and Jem1p.

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