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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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Induction of the  unfolded protein response  pathway. (A and B) Wild-type (RGY131) and mutant  (RGY144, RGY145, RGY324)  yeast strains were grown at  25°C in the absence or presence of 10 μg/ml of tunicamycin (TM). Northern blot analysis of RNA preparations  were performed as described  in Materials and Methods.  (A) KAR2 mRNA, normalized to ACT1 mRNA probed  on the same blot, is expressed  as bars corresponding to the  fold-induction of KAR2  mRNA relative to that expressed by the wild-type  strain. (B) JEM1 mRNA,  normalized to ethidium bromide stained rRNAs, is expressed as bars corresponding to the fold-induction of  JEM1 mRNA relative to  that expressed by the wild-type strain. (C) Kar2p immunoprecipitates from wild-type  (RGY131) and mutant  strains (RGY145, RGY146,  RGY147, and RGY148) that  were radiolabeled for 10 min at 25°C were resolved by PAGE in  SDS. The radioactive band corresponding to Kar2p was quantified and is expressed as fold-induction of Kar2p relative to that  expressed by the wild-type strain at 25°C.
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Figure 7: Induction of the unfolded protein response pathway. (A and B) Wild-type (RGY131) and mutant (RGY144, RGY145, RGY324) yeast strains were grown at 25°C in the absence or presence of 10 μg/ml of tunicamycin (TM). Northern blot analysis of RNA preparations were performed as described in Materials and Methods. (A) KAR2 mRNA, normalized to ACT1 mRNA probed on the same blot, is expressed as bars corresponding to the fold-induction of KAR2 mRNA relative to that expressed by the wild-type strain. (B) JEM1 mRNA, normalized to ethidium bromide stained rRNAs, is expressed as bars corresponding to the fold-induction of JEM1 mRNA relative to that expressed by the wild-type strain. (C) Kar2p immunoprecipitates from wild-type (RGY131) and mutant strains (RGY145, RGY146, RGY147, and RGY148) that were radiolabeled for 10 min at 25°C were resolved by PAGE in SDS. The radioactive band corresponding to Kar2p was quantified and is expressed as fold-induction of Kar2p relative to that expressed by the wild-type strain at 25°C.

Mentions: The accumulation of unfolded proteins in the ER is a signal for increased synthesis of ER chaperones that are regulated by the UPR pathway (Kozutsumi et al., 1988). Increased transcription of the KAR2 gene (Normington et al., 1989; Rose et al., 1989), the promoter of which contains a 22-bp UPR element (UPRE; Mori et al., 1992), is indicative of an increased content of unfolded proteins in the yeast ER. The SCJ1 and JEM1 genes contain UPRE (Blumberg and Silver, 1991), and transcription of both genes is induced by agents that cause protein folding stress (Schlenstedt et al., 1995; Nishikawa and Endo, 1997). Transcription of KAR2 and JEM1 mRNAs was quantified by probing Northern blots of total RNA prepared from yeast cultures grown at 25°C (Fig. 7, A and B). Incubation of wild-type cells for 2 h in the presence of sufficient tunicamycin to completely block assembly of the dolichol-linked oligosaccharide donor for N-linked glycosylation results in a 4.9-fold increase in KAR2 mRNA expression and a 3.5-fold increase in JEM1 mRNA expression. Previous investigators have reported similar extents of KAR2 mRNA induction by tunicamycin treatment (Cox et al., 1993; Baxter et al., 1996). Disruption of the OST3 gene or the SCJ1 gene results in a 2- and a 2.7-fold increase in KAR2 mRNA relative to the wild-type strain, respectively. The combined loss of both Ost3p and Scj1p results in a 3.5-fold induction of KAR2 mRNA, consistent with the view that loss of Scj1p aggravates the protein folding stress caused by hypoglycosylation. In each strain, induction of JEM1 mRNA was slightly lower than induction of KAR2 mRNA (Fig. 7 B). Similar results were obtained for the Δost3, Δost3Δscj1 and Δscj1 strains when induction of the UPR pathway was analyzed by comparing the incorporation of 35S methionine into Kar2p in the wild-type and mutant strains (Fig. 7 C and not shown). Induction of Kar2p was also evaluated in strains in which the JEM1 gene was disrupted (Fig. 7 C). We found that loss of Jem1p has less effect on Kar2p expression than loss of Scj1p. As observed for the Δost3Δscj1 double mutant (Fig. 7 A), disruption of the JEM1 gene in the context of a Δost3 strain or a Δscj1 strain leads to a further increase in Kar2p expression (Fig. 7 C).


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)

Induction of the  unfolded protein response  pathway. (A and B) Wild-type (RGY131) and mutant  (RGY144, RGY145, RGY324)  yeast strains were grown at  25°C in the absence or presence of 10 μg/ml of tunicamycin (TM). Northern blot analysis of RNA preparations  were performed as described  in Materials and Methods.  (A) KAR2 mRNA, normalized to ACT1 mRNA probed  on the same blot, is expressed  as bars corresponding to the  fold-induction of KAR2  mRNA relative to that expressed by the wild-type  strain. (B) JEM1 mRNA,  normalized to ethidium bromide stained rRNAs, is expressed as bars corresponding to the fold-induction of  JEM1 mRNA relative to  that expressed by the wild-type strain. (C) Kar2p immunoprecipitates from wild-type  (RGY131) and mutant  strains (RGY145, RGY146,  RGY147, and RGY148) that  were radiolabeled for 10 min at 25°C were resolved by PAGE in  SDS. The radioactive band corresponding to Kar2p was quantified and is expressed as fold-induction of Kar2p relative to that  expressed by the wild-type strain at 25°C.
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Figure 7: Induction of the unfolded protein response pathway. (A and B) Wild-type (RGY131) and mutant (RGY144, RGY145, RGY324) yeast strains were grown at 25°C in the absence or presence of 10 μg/ml of tunicamycin (TM). Northern blot analysis of RNA preparations were performed as described in Materials and Methods. (A) KAR2 mRNA, normalized to ACT1 mRNA probed on the same blot, is expressed as bars corresponding to the fold-induction of KAR2 mRNA relative to that expressed by the wild-type strain. (B) JEM1 mRNA, normalized to ethidium bromide stained rRNAs, is expressed as bars corresponding to the fold-induction of JEM1 mRNA relative to that expressed by the wild-type strain. (C) Kar2p immunoprecipitates from wild-type (RGY131) and mutant strains (RGY145, RGY146, RGY147, and RGY148) that were radiolabeled for 10 min at 25°C were resolved by PAGE in SDS. The radioactive band corresponding to Kar2p was quantified and is expressed as fold-induction of Kar2p relative to that expressed by the wild-type strain at 25°C.
Mentions: The accumulation of unfolded proteins in the ER is a signal for increased synthesis of ER chaperones that are regulated by the UPR pathway (Kozutsumi et al., 1988). Increased transcription of the KAR2 gene (Normington et al., 1989; Rose et al., 1989), the promoter of which contains a 22-bp UPR element (UPRE; Mori et al., 1992), is indicative of an increased content of unfolded proteins in the yeast ER. The SCJ1 and JEM1 genes contain UPRE (Blumberg and Silver, 1991), and transcription of both genes is induced by agents that cause protein folding stress (Schlenstedt et al., 1995; Nishikawa and Endo, 1997). Transcription of KAR2 and JEM1 mRNAs was quantified by probing Northern blots of total RNA prepared from yeast cultures grown at 25°C (Fig. 7, A and B). Incubation of wild-type cells for 2 h in the presence of sufficient tunicamycin to completely block assembly of the dolichol-linked oligosaccharide donor for N-linked glycosylation results in a 4.9-fold increase in KAR2 mRNA expression and a 3.5-fold increase in JEM1 mRNA expression. Previous investigators have reported similar extents of KAR2 mRNA induction by tunicamycin treatment (Cox et al., 1993; Baxter et al., 1996). Disruption of the OST3 gene or the SCJ1 gene results in a 2- and a 2.7-fold increase in KAR2 mRNA relative to the wild-type strain, respectively. The combined loss of both Ost3p and Scj1p results in a 3.5-fold induction of KAR2 mRNA, consistent with the view that loss of Scj1p aggravates the protein folding stress caused by hypoglycosylation. In each strain, induction of JEM1 mRNA was slightly lower than induction of KAR2 mRNA (Fig. 7 B). Similar results were obtained for the Δost3, Δost3Δscj1 and Δscj1 strains when induction of the UPR pathway was analyzed by comparing the incorporation of 35S methionine into Kar2p in the wild-type and mutant strains (Fig. 7 C and not shown). Induction of Kar2p was also evaluated in strains in which the JEM1 gene was disrupted (Fig. 7 C). We found that loss of Jem1p has less effect on Kar2p expression than loss of Scj1p. As observed for the Δost3Δscj1 double mutant (Fig. 7 A), disruption of the JEM1 gene in the context of a Δost3 strain or a Δscj1 strain leads to a further increase in Kar2p expression (Fig. 7 C).

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