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Functional differences in yeast protein disulfide isomerases.

Nørgaard P, Westphal V, Tachibana C, Alsøe L, Holst B, Winther JR - J. Cell Biol. (2001)

Bottom Line: In several cases, we found that the ability of the PDI1 homologues to restore viability to a pdi1-deleted strain when overexpressed was dependent on the presence of low endogenous levels of one or more of the other homologues.Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification.There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Yeast Genetics, Carlsberg Laboratory, DK-2500 Copenhagen Valby, Denmark.

ABSTRACT
PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several cases, we found that the ability of the PDI1 homologues to restore viability to a pdi1-deleted strain when overexpressed was dependent on the presence of low endogenous levels of one or more of the other homologues. This shows that the homologues are not functionally interchangeable. In fact, Mpd1p was the only homologue capable of carrying out all the essential functions of Pdi1p. Furthermore, the presence of endogenous homologues with a CXXC motif in the thioredoxin-like domain is required for suppression of a pdi1 deletion by EUG1 (which contains two CXXS active site motifs). This underlines the essentiality of protein disulfide isomerase-catalyzed oxidation. Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification. There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains.

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DTT sensitivity of PDI-deficient yeast strains overexpressing PDI1 homologues. Cultures were diluted and applied to SC + 5×Leu + 5×Ade plates buffered to pH 5 and supplemented with different concentrations of DTT. To prevent oxidation of DTT by molecular oxygen, the plates were freshly made and incubated at 30°C in a CO2 atmosphere.
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Figure 3: DTT sensitivity of PDI-deficient yeast strains overexpressing PDI1 homologues. Cultures were diluted and applied to SC + 5×Leu + 5×Ade plates buffered to pH 5 and supplemented with different concentrations of DTT. To prevent oxidation of DTT by molecular oxygen, the plates were freshly made and incubated at 30°C in a CO2 atmosphere.

Mentions: DTT sensitivity is closely linked to the effectiveness of the oxidizing apparatus of the ER (Jämsä et al. 1994; Frand and Kaiser 1998). DTT penetrates the ER and interferes with the formation of disulfide bonds in nascent proteins (Braakman et al. 1991). Mutants of Pdi1p mutated at the two central residues of the active site CGHC sequence furthermore show increased DTT sensitivity (Holst et al. 1997). We were interested in determining to what extent the oxidizing apparatus of the various deletion mutants was affected. A wild-type strain is able to grow in the presence of 5 mM DTT under otherwise normal conditions. To amplify possible differences in DTT sensitivity with respect to growth, we used the mutant PDI1CRRC-SGHS, which had previously been shown to cause increased sensitivity towards DTT (Holst et al. 1997). We found that deletion of all genes encoding the PDI homologues increased the DTT sensitivity of the strain rescued by the mutant gene PDI1CRRC-SGHS, most clearly seen by the difference in growth at 0.5 and 1.0 mM DTT (Fig. 3).


Functional differences in yeast protein disulfide isomerases.

Nørgaard P, Westphal V, Tachibana C, Alsøe L, Holst B, Winther JR - J. Cell Biol. (2001)

DTT sensitivity of PDI-deficient yeast strains overexpressing PDI1 homologues. Cultures were diluted and applied to SC + 5×Leu + 5×Ade plates buffered to pH 5 and supplemented with different concentrations of DTT. To prevent oxidation of DTT by molecular oxygen, the plates were freshly made and incubated at 30°C in a CO2 atmosphere.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195995&req=5

Figure 3: DTT sensitivity of PDI-deficient yeast strains overexpressing PDI1 homologues. Cultures were diluted and applied to SC + 5×Leu + 5×Ade plates buffered to pH 5 and supplemented with different concentrations of DTT. To prevent oxidation of DTT by molecular oxygen, the plates were freshly made and incubated at 30°C in a CO2 atmosphere.
Mentions: DTT sensitivity is closely linked to the effectiveness of the oxidizing apparatus of the ER (Jämsä et al. 1994; Frand and Kaiser 1998). DTT penetrates the ER and interferes with the formation of disulfide bonds in nascent proteins (Braakman et al. 1991). Mutants of Pdi1p mutated at the two central residues of the active site CGHC sequence furthermore show increased DTT sensitivity (Holst et al. 1997). We were interested in determining to what extent the oxidizing apparatus of the various deletion mutants was affected. A wild-type strain is able to grow in the presence of 5 mM DTT under otherwise normal conditions. To amplify possible differences in DTT sensitivity with respect to growth, we used the mutant PDI1CRRC-SGHS, which had previously been shown to cause increased sensitivity towards DTT (Holst et al. 1997). We found that deletion of all genes encoding the PDI homologues increased the DTT sensitivity of the strain rescued by the mutant gene PDI1CRRC-SGHS, most clearly seen by the difference in growth at 0.5 and 1.0 mM DTT (Fig. 3).

Bottom Line: In several cases, we found that the ability of the PDI1 homologues to restore viability to a pdi1-deleted strain when overexpressed was dependent on the presence of low endogenous levels of one or more of the other homologues.Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification.There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Yeast Genetics, Carlsberg Laboratory, DK-2500 Copenhagen Valby, Denmark.

ABSTRACT
PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several cases, we found that the ability of the PDI1 homologues to restore viability to a pdi1-deleted strain when overexpressed was dependent on the presence of low endogenous levels of one or more of the other homologues. This shows that the homologues are not functionally interchangeable. In fact, Mpd1p was the only homologue capable of carrying out all the essential functions of Pdi1p. Furthermore, the presence of endogenous homologues with a CXXC motif in the thioredoxin-like domain is required for suppression of a pdi1 deletion by EUG1 (which contains two CXXS active site motifs). This underlines the essentiality of protein disulfide isomerase-catalyzed oxidation. Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification. There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains.

Show MeSH
Related in: MedlinePlus