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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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Loss of Jem1p aggravates the folding defect of the  Δscj1 mutant. (A) Wild-type (RGY131), Δjem1 (RGY147), and  Δscj1Δjem1 (RGY148) strains bearing pJW373 were radiolabeled  as described in Fig. 3. CPY was immunoprecipitated from glass  bead extracts of radiolabeled cells and resolved by PAGE in  SDS. (B) The radioactive bands corresponding to pug-CPY and  ug-CPY in the gel shown in A were quantified, and the amount of  mature ug-CPY present in each sample was expressed as a percentage of the total amount of pug-CPY and ug-CPY present  at each time point during the chase. (C) Δscj1 (RGY142),  Δost3Δscj1 (RGY144) and Δscj1Δjem1 (RGY148) strains were  transformed with URA3 marked 2μ plasmids that either lack an  insert (yEp352), contain the SCJ1 gene (pPS720) or the JEM1  gene (pMR3270). Yeast strains were grown in liquid minimal media (−uracil) at 25°C and diluted to a cell density of 106 cells/ml.  5-μl aliquots of tenfold serial dilutions were plated on YPDA  plates and incubated at 25 or 37°C for 2 d.
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Figure 4: Loss of Jem1p aggravates the folding defect of the Δscj1 mutant. (A) Wild-type (RGY131), Δjem1 (RGY147), and Δscj1Δjem1 (RGY148) strains bearing pJW373 were radiolabeled as described in Fig. 3. CPY was immunoprecipitated from glass bead extracts of radiolabeled cells and resolved by PAGE in SDS. (B) The radioactive bands corresponding to pug-CPY and ug-CPY in the gel shown in A were quantified, and the amount of mature ug-CPY present in each sample was expressed as a percentage of the total amount of pug-CPY and ug-CPY present at each time point during the chase. (C) Δscj1 (RGY142), Δost3Δscj1 (RGY144) and Δscj1Δjem1 (RGY148) strains were transformed with URA3 marked 2μ plasmids that either lack an insert (yEp352), contain the SCJ1 gene (pPS720) or the JEM1 gene (pMR3270). Yeast strains were grown in liquid minimal media (−uracil) at 25°C and diluted to a cell density of 106 cells/ml. 5-μl aliquots of tenfold serial dilutions were plated on YPDA plates and incubated at 25 or 37°C for 2 d.

Mentions: The intracellular transport of glycosylated and unglycosylated CPY was analyzed in the wild-type, Δjem1 and Δscj1Δjem1 mutant strains to determine whether Jem1p is also involved in protein folding (Fig. 4 A). The time course for conversion of pug-CPY to mature unglycosylated CPY was remarkably similar in the wild-type and Δjem1 strains (Fig. 4 B). However, the transport of unglycosylated CPY to the vacuole was drastically delayed in the Δscj1Δjem1 mutant; after a 60-min chase period, <30% of the total ug-CPY precursor had been converted to the mature form (Fig. 4 B). The 3.2-fold slower transport of ug-CPY in the Δscj1Δjem1 mutant relative to wild-type cells is similar to that observed for several kar2 alleles (te Heesen and Aebi, 1994), and is more severe than that observed for the Δscj1 mutant (Fig. 3 B). Interestingly, the maturation of CPY expressed from the chromosomal PRC1 gene was also delayed in the strain that lacks both DnaJ homologues, since the ER precursor (p1CPY) can still be detected after a 60-min chase in the immunoprecipitates from the Δscj1Δjem1 strain (Fig. 4 A). This delay in transport of wild-type CPY from the ER to the vacuole in the Δscj1Δjem1 mutant was dependent upon the simultaneous expression of the unglycosylated CPY mutant (data not shown). Nontranslocated precursor forms of Kar2p were not detected when Kar2p was immunoprecipitated from pulse-labeled Δscj1Δjem1 cells (not shown), indicating that protein translocation is not perturbed when two of the three DnaJ homologues are absent.


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)

Loss of Jem1p aggravates the folding defect of the  Δscj1 mutant. (A) Wild-type (RGY131), Δjem1 (RGY147), and  Δscj1Δjem1 (RGY148) strains bearing pJW373 were radiolabeled  as described in Fig. 3. CPY was immunoprecipitated from glass  bead extracts of radiolabeled cells and resolved by PAGE in  SDS. (B) The radioactive bands corresponding to pug-CPY and  ug-CPY in the gel shown in A were quantified, and the amount of  mature ug-CPY present in each sample was expressed as a percentage of the total amount of pug-CPY and ug-CPY present  at each time point during the chase. (C) Δscj1 (RGY142),  Δost3Δscj1 (RGY144) and Δscj1Δjem1 (RGY148) strains were  transformed with URA3 marked 2μ plasmids that either lack an  insert (yEp352), contain the SCJ1 gene (pPS720) or the JEM1  gene (pMR3270). Yeast strains were grown in liquid minimal media (−uracil) at 25°C and diluted to a cell density of 106 cells/ml.  5-μl aliquots of tenfold serial dilutions were plated on YPDA  plates and incubated at 25 or 37°C for 2 d.
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Figure 4: Loss of Jem1p aggravates the folding defect of the Δscj1 mutant. (A) Wild-type (RGY131), Δjem1 (RGY147), and Δscj1Δjem1 (RGY148) strains bearing pJW373 were radiolabeled as described in Fig. 3. CPY was immunoprecipitated from glass bead extracts of radiolabeled cells and resolved by PAGE in SDS. (B) The radioactive bands corresponding to pug-CPY and ug-CPY in the gel shown in A were quantified, and the amount of mature ug-CPY present in each sample was expressed as a percentage of the total amount of pug-CPY and ug-CPY present at each time point during the chase. (C) Δscj1 (RGY142), Δost3Δscj1 (RGY144) and Δscj1Δjem1 (RGY148) strains were transformed with URA3 marked 2μ plasmids that either lack an insert (yEp352), contain the SCJ1 gene (pPS720) or the JEM1 gene (pMR3270). Yeast strains were grown in liquid minimal media (−uracil) at 25°C and diluted to a cell density of 106 cells/ml. 5-μl aliquots of tenfold serial dilutions were plated on YPDA plates and incubated at 25 or 37°C for 2 d.
Mentions: The intracellular transport of glycosylated and unglycosylated CPY was analyzed in the wild-type, Δjem1 and Δscj1Δjem1 mutant strains to determine whether Jem1p is also involved in protein folding (Fig. 4 A). The time course for conversion of pug-CPY to mature unglycosylated CPY was remarkably similar in the wild-type and Δjem1 strains (Fig. 4 B). However, the transport of unglycosylated CPY to the vacuole was drastically delayed in the Δscj1Δjem1 mutant; after a 60-min chase period, <30% of the total ug-CPY precursor had been converted to the mature form (Fig. 4 B). The 3.2-fold slower transport of ug-CPY in the Δscj1Δjem1 mutant relative to wild-type cells is similar to that observed for several kar2 alleles (te Heesen and Aebi, 1994), and is more severe than that observed for the Δscj1 mutant (Fig. 3 B). Interestingly, the maturation of CPY expressed from the chromosomal PRC1 gene was also delayed in the strain that lacks both DnaJ homologues, since the ER precursor (p1CPY) can still be detected after a 60-min chase in the immunoprecipitates from the Δscj1Δjem1 strain (Fig. 4 A). This delay in transport of wild-type CPY from the ER to the vacuole in the Δscj1Δjem1 mutant was dependent upon the simultaneous expression of the unglycosylated CPY mutant (data not shown). Nontranslocated precursor forms of Kar2p were not detected when Kar2p was immunoprecipitated from pulse-labeled Δscj1Δjem1 cells (not shown), indicating that protein translocation is not perturbed when two of the three DnaJ homologues are absent.

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
Related in: MedlinePlus