The Contributions of Protein Disulfide Isomerase and Its Homologues to Oxidative Protein Folding in the Yeast Endoplasmic Reticulum

Ruoyu Xiao,Bonney Wilkinson,Anton Solovyov,Jakob R Winther,Arne Holmgren,Johanna Lundström-Ljung,Hiram F Gilbert

doi:10.1074/jbc.m409210200

Abstract

In vitro, protein disulfide isomerase (Pdi1p) introduces disulfides into proteins (oxidase activity) and provides quality control by catalyzing the rearrangement of incorrect disulfides (isomerase activity). Protein disulfide isomerase (PDI) is an essential protein in Saccharomyces cerevisiae, but the contributions of the catalytic activities of PDI to oxidative protein folding in the endoplasmic reticulum (ER) are unclear. Using variants of Pdi1p with impaired oxidase or isomerase activity, we show that isomerase-deficient mutants of PDI support wild-type growth even in a strain in which all of the PDI homologues of the yeast ER have been deleted. Although the oxidase activity of PDI is sufficient for wild-type growth, pulse-chase experiments monitoring the maturation of carboxypeptidase Y reveal that oxidative folding is greatly compromised in mutants that are defective in isomerase activity. Pdi1p and one or more of its ER homologues (Mpd1p, Mpd2p, Eug1p, Eps1p) are required for efficient carboxypeptidase Y maturation. Consistent with its function as a disulfide isomerase in vivo, the active sites of Pdi1p are partially reduced (32 +/- 8%) in vivo. These results suggest that PDI and its ER homologues contribute both oxidase and isomerase activities to the yeast ER. The isomerase activity of PDI can be compromised without affecting growth and viability, implying that yeast proteins that are essential under laboratory conditions may not require efficient disulfide isomerization.

Highlights

Disulfide bonds provide added stability to extracellular proteins by covalently cross-linking two cysteines
To compare the abilities of individual yeast Pdi1p catalytic domains to support growth when expressed from the endogenous PDI1 promoter on a low copy plasmid, genes encoding the a and a؅ domains of yeast protein disulfide isomerase (PDI), full-length yeast PDI and rat PDI were individually introduced into ⌬pdi1 S. cerevisiae using a plasmid shuffling method [12]
The yeast PDIa domain and the individual catalytic domains of rat PDI were not able to support growth when expressed from the PDI1 promoter; they all rescued the lethal phenotype when overexpressed from the PDI1 promoter on a multicopy plasmid (2 ␮m)

Summary

EXPERIMENTAL PROCEDURES

Strains and Plasmids—Saccharomyces cerevisiae strains with complete deletions of PDI1 were obtained from Ron Raines (University of Wisconsin, Madison, WI) [12] and from Norgaard et al [18]. The protein was resuspended in 5 mM gel-filtered Mal-PEG in non-reducing SDS sample buffer (3% SDS, 0.2 M Tris-HCl, pH 8, glycerol, bromphenol blue) and incubated for 30 min at room temperature quenched by the addition of DTT to a final concentration of 50 mM. Each sample contained 4.5 mM cCMP, 1 mM GSH, 0.2 mM GSSG, 100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 8 ␮M reduced RNase, and 0 –9 ␮M yeast PDI or Disulfide Formation by PDI. The insoluble material was washed with ice-cold acetone four times, and the pellets were resuspended in IP buffer (50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 1% Triton, 0.2% SDS, and 150 mM NaCl), boiled for 5 min, and incubated with Pansorbin (50 ␮l) (Calbiochem) for 20 min. Screens were scanned using StormScan scanners to detect radiolabeled CPY bands

RESULTS

TABLE I Rescue of PDI deficiencies by expression of various PDIs

Introduced gene

DISCUSSION