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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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Characterization of  ost1-6, a temperature-sensitive allele of the OST1 gene.  (A) Growth phenotype of  the ost1-6 mutant. Yeast  strains (RGY140, RGY140  [pRS316-OST1], RGY141,  and RGY142) were streaked  on YPDA-agar and incubated for 2 d at the indicated  temperatures. (B) Hypoglycosylation of CPY by the ost1-6  mutant. CPY immunoprecipitates from glass bead extracts of yeast strains radiolabeled for 1 h at 25°C were resolved by PAGE in SDS. Fully glycosylated vacuolar  CPY and hypoglycosylated variants of CPY lacking between 1 and 4 N-linked oligosaccharides are indicated by the labeled arrows designated −1 through −4.
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Figure 1: Characterization of ost1-6, a temperature-sensitive allele of the OST1 gene. (A) Growth phenotype of the ost1-6 mutant. Yeast strains (RGY140, RGY140 [pRS316-OST1], RGY141, and RGY142) were streaked on YPDA-agar and incubated for 2 d at the indicated temperatures. (B) Hypoglycosylation of CPY by the ost1-6 mutant. CPY immunoprecipitates from glass bead extracts of yeast strains radiolabeled for 1 h at 25°C were resolved by PAGE in SDS. Fully glycosylated vacuolar CPY and hypoglycosylated variants of CPY lacking between 1 and 4 N-linked oligosaccharides are indicated by the labeled arrows designated −1 through −4.

Mentions: The initial ost1-6 strain was backcrossed several times with RGY131 to minimize the effect of strain background on the subsequent analysis, and to separate the Δscj1 and ost1-6 mutations. In this new genetic background, the resulting ost1-6 strain displays a temperature-sensitive phenotype (Fig. 1 A). Previously isolated ost1 mutants have reduced OST activity as detected by pleiotropic hypoglycosylation of N-linked glycoproteins synthesized in vivo at both the permissive (25°C) and restrictive (37°C) temperatures (Silberstein et al., 1995). Biosynthesis of the soluble vacuolar glycoprotein CPY by wild-type, Δscj1, ost1-6, and ost1-6 cells complemented with pRS316-OST1 was analyzed to determine whether the ost1-6 mutant has reduced OST activity at the permissive temperature. CPY is synthesized as a proenzyme that acquires four N-linked oligosaccharides upon translocation into the lumen of the endoplasmic reticulum. The 67-kD ER form of proCPY (p1CPY) is transported to the Golgi complex, where the core oligosaccharides are elongated by the addition of mannose residues to yield the 69-kD Golgi form of proCPY (p2CPY). Upon transport to the vacuole, proteolytic removal of an 8-kD propeptide generates the mature 61-kD vacuolar form of CPY. CPY was immunoprecipitated from yeast cultures that were radiolabeled with 35S methionine for 1 h at 25°C (Fig. 1 B). As expected, the predominant form of CPY synthesized by the wild-type and Δscj1 mutant has four N-linked oligosaccharides. In addition to fully glycosylated CPY, the ost1-6 mutant strain synthesized hypoglycosylated variants of CPY that lack between one and three oligosaccharides (Fig. 1 B, −1, −2, and −3 forms of CPY). Notably, the synthetic lethal phenotype, the temperature sensitive growth defect (Fig. 1 A), and the glycosylation defect of the ost1-6 mutant (Fig. 1 B) are corrected by expression of Ost1p from a centromeric plasmid. From the preceding results, we conclude that the temperature-sensitive phenotype of the ost1-6 allele is due to reduced levels of N-glycosylation caused by a mutation in OST1, the α subunit of the OST complex.


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)

Characterization of  ost1-6, a temperature-sensitive allele of the OST1 gene.  (A) Growth phenotype of  the ost1-6 mutant. Yeast  strains (RGY140, RGY140  [pRS316-OST1], RGY141,  and RGY142) were streaked  on YPDA-agar and incubated for 2 d at the indicated  temperatures. (B) Hypoglycosylation of CPY by the ost1-6  mutant. CPY immunoprecipitates from glass bead extracts of yeast strains radiolabeled for 1 h at 25°C were resolved by PAGE in SDS. Fully glycosylated vacuolar  CPY and hypoglycosylated variants of CPY lacking between 1 and 4 N-linked oligosaccharides are indicated by the labeled arrows designated −1 through −4.
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Related In: Results  -  Collection

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Figure 1: Characterization of ost1-6, a temperature-sensitive allele of the OST1 gene. (A) Growth phenotype of the ost1-6 mutant. Yeast strains (RGY140, RGY140 [pRS316-OST1], RGY141, and RGY142) were streaked on YPDA-agar and incubated for 2 d at the indicated temperatures. (B) Hypoglycosylation of CPY by the ost1-6 mutant. CPY immunoprecipitates from glass bead extracts of yeast strains radiolabeled for 1 h at 25°C were resolved by PAGE in SDS. Fully glycosylated vacuolar CPY and hypoglycosylated variants of CPY lacking between 1 and 4 N-linked oligosaccharides are indicated by the labeled arrows designated −1 through −4.
Mentions: The initial ost1-6 strain was backcrossed several times with RGY131 to minimize the effect of strain background on the subsequent analysis, and to separate the Δscj1 and ost1-6 mutations. In this new genetic background, the resulting ost1-6 strain displays a temperature-sensitive phenotype (Fig. 1 A). Previously isolated ost1 mutants have reduced OST activity as detected by pleiotropic hypoglycosylation of N-linked glycoproteins synthesized in vivo at both the permissive (25°C) and restrictive (37°C) temperatures (Silberstein et al., 1995). Biosynthesis of the soluble vacuolar glycoprotein CPY by wild-type, Δscj1, ost1-6, and ost1-6 cells complemented with pRS316-OST1 was analyzed to determine whether the ost1-6 mutant has reduced OST activity at the permissive temperature. CPY is synthesized as a proenzyme that acquires four N-linked oligosaccharides upon translocation into the lumen of the endoplasmic reticulum. The 67-kD ER form of proCPY (p1CPY) is transported to the Golgi complex, where the core oligosaccharides are elongated by the addition of mannose residues to yield the 69-kD Golgi form of proCPY (p2CPY). Upon transport to the vacuole, proteolytic removal of an 8-kD propeptide generates the mature 61-kD vacuolar form of CPY. CPY was immunoprecipitated from yeast cultures that were radiolabeled with 35S methionine for 1 h at 25°C (Fig. 1 B). As expected, the predominant form of CPY synthesized by the wild-type and Δscj1 mutant has four N-linked oligosaccharides. In addition to fully glycosylated CPY, the ost1-6 mutant strain synthesized hypoglycosylated variants of CPY that lack between one and three oligosaccharides (Fig. 1 B, −1, −2, and −3 forms of CPY). Notably, the synthetic lethal phenotype, the temperature sensitive growth defect (Fig. 1 A), and the glycosylation defect of the ost1-6 mutant (Fig. 1 B) are corrected by expression of Ost1p from a centromeric plasmid. From the preceding results, we conclude that the temperature-sensitive phenotype of the ost1-6 allele is due to reduced levels of N-glycosylation caused by a mutation in OST1, the α subunit of the OST complex.

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