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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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Intracellular folding and maturation of CPY followed by immunoprecipitation from pulse-labeled cells separated on SDS-PAGE. Cells were labeled for 15 min and chased for 0, 15, and 60 min. (A) A Δpdi1 strain rescued by the indicated genes. (B) Comparison of a Δpdi1 and a Δpdi1 Δmpd1 Δmpd2 Δeug1 Δeps1 strain containing plasmids with PDI1 or MPD1.
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Figure 4: Intracellular folding and maturation of CPY followed by immunoprecipitation from pulse-labeled cells separated on SDS-PAGE. Cells were labeled for 15 min and chased for 0, 15, and 60 min. (A) A Δpdi1 strain rescued by the indicated genes. (B) Comparison of a Δpdi1 and a Δpdi1 Δmpd1 Δmpd2 Δeug1 Δeps1 strain containing plasmids with PDI1 or MPD1.

Mentions: We followed the maturation of proCPY to CPY in strains depleted for PDIs. Cells were labeled with [35S]methionine for 15 min and chased for 0, 15, and 60 min. After immunoprecipitation with CPY antibody, the samples were subjected to SDS-PAGE and analyzed using a PhosphorImager. As seen in Fig. 4 A, the rate of proCPY folding was strongly dependent on the gene that was used to rescue the pdi1 deletion. The half-time of proCPY maturation in the PDI1-complemented strain is 5–10 min, whereas the half time increased to ∼30 min in strains rescued by overexpression of MPD1. When the pdi1 deletion is rescued by overexpression of EUG1, proCPY maturation is almost arrested, accompanied by accumulation of the p1 ER form of proCPY. This effect is even more pronounced in the strain rescued by overexpression of MPD2. Deletion of the genes encoding the various homologues had no, or very little, effect on the rate of proCPY maturation as long as the strains were complemented by PDI1 (Fig. 4 B), indicating that the Pdi1p homologues do not normally make a significant contribution to proCPY folding. However, the homologues do recognize proCPY as a substrate in the absence of Pdi1p, because maturation of proCPY takes place, although at a reduced rate.


Functional differences in yeast protein disulfide isomerases.

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

Intracellular folding and maturation of CPY followed by immunoprecipitation from pulse-labeled cells separated on SDS-PAGE. Cells were labeled for 15 min and chased for 0, 15, and 60 min. (A) A Δpdi1 strain rescued by the indicated genes. (B) Comparison of a Δpdi1 and a Δpdi1 Δmpd1 Δmpd2 Δeug1 Δeps1 strain containing plasmids with PDI1 or MPD1.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Intracellular folding and maturation of CPY followed by immunoprecipitation from pulse-labeled cells separated on SDS-PAGE. Cells were labeled for 15 min and chased for 0, 15, and 60 min. (A) A Δpdi1 strain rescued by the indicated genes. (B) Comparison of a Δpdi1 and a Δpdi1 Δmpd1 Δmpd2 Δeug1 Δeps1 strain containing plasmids with PDI1 or MPD1.
Mentions: We followed the maturation of proCPY to CPY in strains depleted for PDIs. Cells were labeled with [35S]methionine for 15 min and chased for 0, 15, and 60 min. After immunoprecipitation with CPY antibody, the samples were subjected to SDS-PAGE and analyzed using a PhosphorImager. As seen in Fig. 4 A, the rate of proCPY folding was strongly dependent on the gene that was used to rescue the pdi1 deletion. The half-time of proCPY maturation in the PDI1-complemented strain is 5–10 min, whereas the half time increased to ∼30 min in strains rescued by overexpression of MPD1. When the pdi1 deletion is rescued by overexpression of EUG1, proCPY maturation is almost arrested, accompanied by accumulation of the p1 ER form of proCPY. This effect is even more pronounced in the strain rescued by overexpression of MPD2. Deletion of the genes encoding the various homologues had no, or very little, effect on the rate of proCPY maturation as long as the strains were complemented by PDI1 (Fig. 4 B), indicating that the Pdi1p homologues do not normally make a significant contribution to proCPY folding. However, the homologues do recognize proCPY as a substrate in the absence of Pdi1p, because maturation of proCPY takes place, although at a reduced rate.

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