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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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Growth of wild-type (RGY131) and  mutant strains (RGY324, RGY144, RGY145,  RGY147, RGY146) on media containing tunicamycin, β-mercaptoethanol, Calcofluor  White, or hygromycin B. Yeast strains were  grown in liquid YPDA medium at 25°C and  diluted to a cell density of 106 cells/ml. 5-μl  Aliquots of 10-fold serial dilutions were  plated on YPDA-agar or on plates with the  same medium containing drugs at the indicated concentrations. YPDA plates without  drug were incubated at 25°C for 2 d. Plates  containing tunicamycin, β-mercaptoethanol,  Calcofluor White and hygromycin B were incubated in the dark for 5–6 d at 25°C.
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Figure 6: Growth of wild-type (RGY131) and mutant strains (RGY324, RGY144, RGY145, RGY147, RGY146) on media containing tunicamycin, β-mercaptoethanol, Calcofluor White, or hygromycin B. Yeast strains were grown in liquid YPDA medium at 25°C and diluted to a cell density of 106 cells/ml. 5-μl Aliquots of 10-fold serial dilutions were plated on YPDA-agar or on plates with the same medium containing drugs at the indicated concentrations. YPDA plates without drug were incubated at 25°C for 2 d. Plates containing tunicamycin, β-mercaptoethanol, Calcofluor White and hygromycin B were incubated in the dark for 5–6 d at 25°C.

Mentions: The synthetic growth defect produced by the combination of a reduction in OST activity and a allele of Scj1p is most readily explained by a protein folding defect that is ultimately responsible for reduced exit of hypoglycosylated proteins from the ER. To determine whether loss of Scj1p or Jem1p makes yeast cells more sensitive to the stress caused by accumulation of unfolded proteins in the ER, the growth of wild-type and mutant strains was compared on plates that contained sublethal concentrations of tunicamycin or β-mercaptoethanol (Fig. 6). All the strains analyzed grew at wild-type rates on YPDA plates incubated at 25°C. The Δscj1 and the Δost3Δscj1 mutant strains showed reduced growth rates compared with the wild-type strain when the YPDA plates contained tunicamycin at a concentration of 1 μg/ml. A less-pronounced sensitivity to tunicamycin, as detected by a slight reduction in colony size, was observed for the Δost3Δjem1 strain relative to the Δost3 strain. The Δost3 strain is slightly more resistant to tunicamycin than the wild-type strain. Resistance to low concentrations of tunicamycin is also a property of the ost1 and ost2 mutants; the degree of resistance is roughly proportional to the severity of the glycosylation defect (Silberstein and Gilmore, unpublished observation). The Δost3 and the Δjem1 mutants were also less sensitive to β-mercaptoethanol than the wild-type strain or the other mutants. Notably, RER proteins involved in disulfide bond formation including protein disulfide isomerase and Ero1p are regulated by the UPR pathway (Frand and Kaiser, 1998). Whereas mutations in the OST result in increased, rather than decreased, resistance to agents that promote protein folding stress, we consider it unlikely that the OST has an intrinsic role as a chaperone. In contrast, the Δost3 Δscj1 and Δost3Δjem1 double mutant strains showed a more pronounced growth defect than the wild-type and Δscj1 strains in plates containing β-mercaptoethanol. Apparently, the combination of hypoglycosylation stress and redox stress is not tolerated in cells that are deficient in Scj1p or 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)

Growth of wild-type (RGY131) and  mutant strains (RGY324, RGY144, RGY145,  RGY147, RGY146) on media containing tunicamycin, β-mercaptoethanol, Calcofluor  White, or hygromycin B. Yeast strains were  grown in liquid YPDA medium at 25°C and  diluted to a cell density of 106 cells/ml. 5-μl  Aliquots of 10-fold serial dilutions were  plated on YPDA-agar or on plates with the  same medium containing drugs at the indicated concentrations. YPDA plates without  drug were incubated at 25°C for 2 d. Plates  containing tunicamycin, β-mercaptoethanol,  Calcofluor White and hygromycin B were incubated in the dark for 5–6 d at 25°C.
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Related In: Results  -  Collection

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Figure 6: Growth of wild-type (RGY131) and mutant strains (RGY324, RGY144, RGY145, RGY147, RGY146) on media containing tunicamycin, β-mercaptoethanol, Calcofluor White, or hygromycin B. Yeast strains were grown in liquid YPDA medium at 25°C and diluted to a cell density of 106 cells/ml. 5-μl Aliquots of 10-fold serial dilutions were plated on YPDA-agar or on plates with the same medium containing drugs at the indicated concentrations. YPDA plates without drug were incubated at 25°C for 2 d. Plates containing tunicamycin, β-mercaptoethanol, Calcofluor White and hygromycin B were incubated in the dark for 5–6 d at 25°C.
Mentions: The synthetic growth defect produced by the combination of a reduction in OST activity and a allele of Scj1p is most readily explained by a protein folding defect that is ultimately responsible for reduced exit of hypoglycosylated proteins from the ER. To determine whether loss of Scj1p or Jem1p makes yeast cells more sensitive to the stress caused by accumulation of unfolded proteins in the ER, the growth of wild-type and mutant strains was compared on plates that contained sublethal concentrations of tunicamycin or β-mercaptoethanol (Fig. 6). All the strains analyzed grew at wild-type rates on YPDA plates incubated at 25°C. The Δscj1 and the Δost3Δscj1 mutant strains showed reduced growth rates compared with the wild-type strain when the YPDA plates contained tunicamycin at a concentration of 1 μg/ml. A less-pronounced sensitivity to tunicamycin, as detected by a slight reduction in colony size, was observed for the Δost3Δjem1 strain relative to the Δost3 strain. The Δost3 strain is slightly more resistant to tunicamycin than the wild-type strain. Resistance to low concentrations of tunicamycin is also a property of the ost1 and ost2 mutants; the degree of resistance is roughly proportional to the severity of the glycosylation defect (Silberstein and Gilmore, unpublished observation). The Δost3 and the Δjem1 mutants were also less sensitive to β-mercaptoethanol than the wild-type strain or the other mutants. Notably, RER proteins involved in disulfide bond formation including protein disulfide isomerase and Ero1p are regulated by the UPR pathway (Frand and Kaiser, 1998). Whereas mutations in the OST result in increased, rather than decreased, resistance to agents that promote protein folding stress, we consider it unlikely that the OST has an intrinsic role as a chaperone. In contrast, the Δost3 Δscj1 and Δost3Δjem1 double mutant strains showed a more pronounced growth defect than the wild-type and Δscj1 strains in plates containing β-mercaptoethanol. Apparently, the combination of hypoglycosylation stress and redox stress is not tolerated in cells that are deficient in Scj1p or 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.

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