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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 and N-linked glycosylation of the Δost3Δscj1  mutant. (A) Severe growth defect of the Δost3Δscj1 mutant at  37°C. Yeast strains (RGY143A, RGY143B, RGY143C, and  RGY143D) were grown in liquid YPDA medium at 25°C and diluted to a density of 106 cells/ml. 5-μl aliquots of 10-fold serial dilutions of the cultures were plated on YPD-agar and incubated at  25 and 37°C for 2 d. (B) Glycosylation of CPY in vivo. CPY was  immunoprecipitated from glass bead extracts of yeast cultures radiolabeled for 10 min at 25°C and then chased for 0 or 15 min.  The ER form (p1CPY), the Golgi form (p2CPY), and fully glycosylated vacuolar CPY were resolved by PAGE in SDS and are  designated by labeled arrows. The migration positions corresponding to hypoglycosylated variants of CPY lacking between 1  and 4 N-linked oligosaccharides are indicated.
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Figure 2: Growth and N-linked glycosylation of the Δost3Δscj1 mutant. (A) Severe growth defect of the Δost3Δscj1 mutant at 37°C. Yeast strains (RGY143A, RGY143B, RGY143C, and RGY143D) were grown in liquid YPDA medium at 25°C and diluted to a density of 106 cells/ml. 5-μl aliquots of 10-fold serial dilutions of the cultures were plated on YPD-agar and incubated at 25 and 37°C for 2 d. (B) Glycosylation of CPY in vivo. CPY was immunoprecipitated from glass bead extracts of yeast cultures radiolabeled for 10 min at 25°C and then chased for 0 or 15 min. The ER form (p1CPY), the Golgi form (p2CPY), and fully glycosylated vacuolar CPY were resolved by PAGE in SDS and are designated by labeled arrows. The migration positions corresponding to hypoglycosylated variants of CPY lacking between 1 and 4 N-linked oligosaccharides are indicated.

Mentions: We postulated that the synthetic lethal phenotype produced by the combination of the Δscj1 and ost1-6 mutations was not caused by an exaggeration of the glycosylation defect inherent to ost1-6, as glycosylation of CPY in the Δscj1 strain is indistinguishable from that of the wild-type strain. However, given that Scj1p is proposed to cooperate with the ER chaperone Kar2p (Schlenstedt et al., 1995), it is formally possible that folding or oligomeric assembly of the mutant form of Ost1p is impaired in a Δscj1 strain thereby causing an indirect reduction in OST activity. To address this possibility, the Δscj1 strain was crossed to a milder OST mutant caused by disruption of the nonessential OST3 gene encoding the 34-kD subunit of the OST. We selected the Δost3 mutant for this analysis because the strain grows at a wild-type rate at 25, 30, and 37°C (Karaoglu et al., 1995a). Moreover, immunoprecipitation experiments from strains that express an epitope-tagged OST subunit (Stt3p) have revealed that the integrity and stability of the OST complex is not compromised in yeast cells that do not express Ost3p (Karaoglu et al., 1997). After sporulation of the Δost3Δscj1 diploid, dissection of tetrads yielded four viable colonies at 25°C from tetratype asci. As reported previously (Blumberg and Silver, 1991; Karaoglu et al., 1995a), the Δost3 and Δscj1 mutants grow at wild-type rates on YPD plates incubated at 25°C or 37°C. In contrast, the Δost3Δscj1 double mutant has a severe growth defect at 37°C, but not at 25°C (Fig. 2 A).


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 and N-linked glycosylation of the Δost3Δscj1  mutant. (A) Severe growth defect of the Δost3Δscj1 mutant at  37°C. Yeast strains (RGY143A, RGY143B, RGY143C, and  RGY143D) were grown in liquid YPDA medium at 25°C and diluted to a density of 106 cells/ml. 5-μl aliquots of 10-fold serial dilutions of the cultures were plated on YPD-agar and incubated at  25 and 37°C for 2 d. (B) Glycosylation of CPY in vivo. CPY was  immunoprecipitated from glass bead extracts of yeast cultures radiolabeled for 10 min at 25°C and then chased for 0 or 15 min.  The ER form (p1CPY), the Golgi form (p2CPY), and fully glycosylated vacuolar CPY were resolved by PAGE in SDS and are  designated by labeled arrows. The migration positions corresponding to hypoglycosylated variants of CPY lacking between 1  and 4 N-linked oligosaccharides are indicated.
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Related In: Results  -  Collection

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Figure 2: Growth and N-linked glycosylation of the Δost3Δscj1 mutant. (A) Severe growth defect of the Δost3Δscj1 mutant at 37°C. Yeast strains (RGY143A, RGY143B, RGY143C, and RGY143D) were grown in liquid YPDA medium at 25°C and diluted to a density of 106 cells/ml. 5-μl aliquots of 10-fold serial dilutions of the cultures were plated on YPD-agar and incubated at 25 and 37°C for 2 d. (B) Glycosylation of CPY in vivo. CPY was immunoprecipitated from glass bead extracts of yeast cultures radiolabeled for 10 min at 25°C and then chased for 0 or 15 min. The ER form (p1CPY), the Golgi form (p2CPY), and fully glycosylated vacuolar CPY were resolved by PAGE in SDS and are designated by labeled arrows. The migration positions corresponding to hypoglycosylated variants of CPY lacking between 1 and 4 N-linked oligosaccharides are indicated.
Mentions: We postulated that the synthetic lethal phenotype produced by the combination of the Δscj1 and ost1-6 mutations was not caused by an exaggeration of the glycosylation defect inherent to ost1-6, as glycosylation of CPY in the Δscj1 strain is indistinguishable from that of the wild-type strain. However, given that Scj1p is proposed to cooperate with the ER chaperone Kar2p (Schlenstedt et al., 1995), it is formally possible that folding or oligomeric assembly of the mutant form of Ost1p is impaired in a Δscj1 strain thereby causing an indirect reduction in OST activity. To address this possibility, the Δscj1 strain was crossed to a milder OST mutant caused by disruption of the nonessential OST3 gene encoding the 34-kD subunit of the OST. We selected the Δost3 mutant for this analysis because the strain grows at a wild-type rate at 25, 30, and 37°C (Karaoglu et al., 1995a). Moreover, immunoprecipitation experiments from strains that express an epitope-tagged OST subunit (Stt3p) have revealed that the integrity and stability of the OST complex is not compromised in yeast cells that do not express Ost3p (Karaoglu et al., 1997). After sporulation of the Δost3Δscj1 diploid, dissection of tetrads yielded four viable colonies at 25°C from tetratype asci. As reported previously (Blumberg and Silver, 1991; Karaoglu et al., 1995a), the Δost3 and Δscj1 mutants grow at wild-type rates on YPD plates incubated at 25°C or 37°C. In contrast, the Δost3Δscj1 double mutant has a severe growth defect at 37°C, but not at 25°C (Fig. 2 A).

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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