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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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Related in: MedlinePlus

Western blot showing the steady state pool of CPY. (A) A wild-type and a Δpdi1 [EUG1] strain. Cells were lysed in the presence or absence of 20 mM AMS. In the third lane, the wild type was treated with 40 mM DTT before lysis and AMS modification. (B) Western blot showing glycosylation defects of CPY in a strain with Mpd1p as only PDI source. A wild-type and a Δpdi1 Δeug1 Δmpd1 Δmpd2 Δeps1 [MPD1] strain show differences in the mobility of the various forms of CPY.
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Figure 5: Western blot showing the steady state pool of CPY. (A) A wild-type and a Δpdi1 [EUG1] strain. Cells were lysed in the presence or absence of 20 mM AMS. In the third lane, the wild type was treated with 40 mM DTT before lysis and AMS modification. (B) Western blot showing glycosylation defects of CPY in a strain with Mpd1p as only PDI source. A wild-type and a Δpdi1 Δeug1 Δmpd1 Δmpd2 Δeps1 [MPD1] strain show differences in the mobility of the various forms of CPY.

Mentions: As Eug1p is not on its own able to carry out oxidation reactions, oxidation should be less efficient in a Δpdi1 strain complemented by EUG1. We tested this assumption by looking at the redox state of proCPY under steady state. Extracts were made in the presence or absence of the thiol modifying agent AMS. AMS modifies free sulfhydryl groups only, and the modification causes a mobility shift on SDS-PAGE due to the bulkiness of the AMS group. The cell extracts were separated on an SDS-PAGE, blotted onto a nitrocellulose membrane, and probed with CPY antibodies. As seen in Fig. 5 A, proCPY in a wild-type strain is not modified by AMS, while treatment of the cells with DTT before the AMS modification causes a large mobility shift on the gel. This corresponds to modification of all eleven cysteines in the fully reduced proCPY. proCPY in the Δpdi1 [EUG1] strain is partially AMS modifiable, suggesting that a significant portion of the proCPY is not completely oxidized at steady state. This indicates that oxidation is in fact compromised in the EUG1 complemented strain.


Functional differences in yeast protein disulfide isomerases.

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

Western blot showing the steady state pool of CPY. (A) A wild-type and a Δpdi1 [EUG1] strain. Cells were lysed in the presence or absence of 20 mM AMS. In the third lane, the wild type was treated with 40 mM DTT before lysis and AMS modification. (B) Western blot showing glycosylation defects of CPY in a strain with Mpd1p as only PDI source. A wild-type and a Δpdi1 Δeug1 Δmpd1 Δmpd2 Δeps1 [MPD1] strain show differences in the mobility of the various forms of CPY.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Western blot showing the steady state pool of CPY. (A) A wild-type and a Δpdi1 [EUG1] strain. Cells were lysed in the presence or absence of 20 mM AMS. In the third lane, the wild type was treated with 40 mM DTT before lysis and AMS modification. (B) Western blot showing glycosylation defects of CPY in a strain with Mpd1p as only PDI source. A wild-type and a Δpdi1 Δeug1 Δmpd1 Δmpd2 Δeps1 [MPD1] strain show differences in the mobility of the various forms of CPY.
Mentions: As Eug1p is not on its own able to carry out oxidation reactions, oxidation should be less efficient in a Δpdi1 strain complemented by EUG1. We tested this assumption by looking at the redox state of proCPY under steady state. Extracts were made in the presence or absence of the thiol modifying agent AMS. AMS modifies free sulfhydryl groups only, and the modification causes a mobility shift on SDS-PAGE due to the bulkiness of the AMS group. The cell extracts were separated on an SDS-PAGE, blotted onto a nitrocellulose membrane, and probed with CPY antibodies. As seen in Fig. 5 A, proCPY in a wild-type strain is not modified by AMS, while treatment of the cells with DTT before the AMS modification causes a large mobility shift on the gel. This corresponds to modification of all eleven cysteines in the fully reduced proCPY. proCPY in the Δpdi1 [EUG1] strain is partially AMS modifiable, suggesting that a significant portion of the proCPY is not completely oxidized at steady state. This indicates that oxidation is in fact compromised in the EUG1 complemented strain.

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