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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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Transport of unglycosylated CPY from the ER  to the vacuole is delayed in  a Δscj1 mutant. (A) Wild-type (RGY141) and Δscj1  (RGY142) strains bearing  pJW373 (encoding ug-CPY)  were radiolabeled for 10 min  at 25°C and chased for the indicated times. CPY immunoprecipitates were 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 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 3: Transport of unglycosylated CPY from the ER to the vacuole is delayed in a Δscj1 mutant. (A) Wild-type (RGY141) and Δscj1 (RGY142) strains bearing pJW373 (encoding ug-CPY) were radiolabeled for 10 min at 25°C and chased for the indicated times. CPY immunoprecipitates were 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 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: CPY was immunoprecipitated from yeast cultures that were pulse-labeled for 10 min at 25°C, and then chased for the indicated times (Fig. 3 A). The ER and Golgi precursor forms of wild-type CPY (p1CPY, p2CPY) and of the mutant CPY (pug-CPY) can be readily distinguished from the mature vacuolar forms of wild-type CPY and mutant ug-CPY by their different mobilities after PAGE in SDS. After a 10-min pulse label, the ER and Golgi precursors are the predominant forms of CPY and ug-CPY detected in both yeast strains. Conversion of the precursors to the more rapidly migrating mature forms during the chase period can be used to estimate the rate at which folded CPY exits the ER. The effect of the Scj1 protein on transport was quantified by determining the percentage of mature CPY (Fig. 3 B, left) and mature ug-CPY (Fig. 3 B, right) during the 1-h chase incubation. During the chase period, similar percentages of mature CPY were obtained from the wild-type and Δscj1 strains, suggesting that Scj1p is not required for a rate-limiting step in transport of fully glycosylated CPY from the ER to the vacuole (Fig. 3 B). Consistent with previous reports (Winther et al., 1991), transport of unglycosylated CPY from the ER to the vacuole was slightly delayed compared with transport of wild-type CPY (Fig. 3 B, compare solid bars in left and right panels). In contrast to what was observed for wild-type CPY, the Δscj1 yeast cells were found to transport unglycosylated CPY to the vacuole 1.7-fold slower than the wild-type strain (Fig. 3 B, right). Loss of Scj1p delays the folding of hypoglycosylated forms of CPY, albeit to a lesser extent than reported previously for several kar2 alleles (te Heesen and Aebi, 1994).


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)

Transport of unglycosylated CPY from the ER  to the vacuole is delayed in  a Δscj1 mutant. (A) Wild-type (RGY141) and Δscj1  (RGY142) strains bearing  pJW373 (encoding ug-CPY)  were radiolabeled for 10 min  at 25°C and chased for the indicated times. CPY immunoprecipitates were 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 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 3: Transport of unglycosylated CPY from the ER to the vacuole is delayed in a Δscj1 mutant. (A) Wild-type (RGY141) and Δscj1 (RGY142) strains bearing pJW373 (encoding ug-CPY) were radiolabeled for 10 min at 25°C and chased for the indicated times. CPY immunoprecipitates were 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 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: CPY was immunoprecipitated from yeast cultures that were pulse-labeled for 10 min at 25°C, and then chased for the indicated times (Fig. 3 A). The ER and Golgi precursor forms of wild-type CPY (p1CPY, p2CPY) and of the mutant CPY (pug-CPY) can be readily distinguished from the mature vacuolar forms of wild-type CPY and mutant ug-CPY by their different mobilities after PAGE in SDS. After a 10-min pulse label, the ER and Golgi precursors are the predominant forms of CPY and ug-CPY detected in both yeast strains. Conversion of the precursors to the more rapidly migrating mature forms during the chase period can be used to estimate the rate at which folded CPY exits the ER. The effect of the Scj1 protein on transport was quantified by determining the percentage of mature CPY (Fig. 3 B, left) and mature ug-CPY (Fig. 3 B, right) during the 1-h chase incubation. During the chase period, similar percentages of mature CPY were obtained from the wild-type and Δscj1 strains, suggesting that Scj1p is not required for a rate-limiting step in transport of fully glycosylated CPY from the ER to the vacuole (Fig. 3 B). Consistent with previous reports (Winther et al., 1991), transport of unglycosylated CPY from the ER to the vacuole was slightly delayed compared with transport of wild-type CPY (Fig. 3 B, compare solid bars in left and right panels). In contrast to what was observed for wild-type CPY, the Δscj1 yeast cells were found to transport unglycosylated CPY to the vacuole 1.7-fold slower than the wild-type strain (Fig. 3 B, right). Loss of Scj1p delays the folding of hypoglycosylated forms of CPY, albeit to a lesser extent than reported previously for several kar2 alleles (te Heesen and Aebi, 1994).

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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