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© The Rockefeller University Press, 0021-9525/1997//1229 $5.00
The Journal of Cell Biology, Volume 138, Number 6, , 1997 1229-1238

Article

Active Site Mutations in Yeast Protein Disulfide Isomerase Cause Dithiothreitol Sensitivity and a Reduced Rate of Protein Folding in the Endoplasmic Reticulum



Bjørn Holst*, Christine Tachibana*,{ddagger}, and Jakob R. Winther*

* Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark; and {ddagger} Department of Biochemistry and Molecular Biology, Penn State University, University Park, Pennsylvania 16802

Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a', each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS. Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a' domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC active site were randomly mutagenized. The resulting mutant PDIs were ranked by their growth phenotype on medium containing increasing concentrations of DTT. The rate of CPY folding in the mutants showed the same ranking as the DTT sensitivity, suggesting that the oxidative power of PDI is an important factor in folding in vivo. Mutants with a PDI that cannot perform oxidation reactions on its own (CGHS) had a strongly reduced growth rate. The growth rates, however, did not correlate with CPY folding, suggesting that the protein(s) required for optimal growth are dependent on PDI for oxidation. pdi1-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth.

Abbreviations used in this paper: CPY, carboxypeptidase Y; GSSG, glutathione disulfide; PDI, protein disulfide isomerase.

Address all correspondence to Dr. Jakob R. Winther, Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark. Tel: +45 3327 5282. Fax: +45 3327 4765. E-mail: jrw{at}crc.dk

C. Tachibana was supported in part by the PSU Office of International Cooperative Programs Global Fund.

2. We have adopted a nomenclature for mutants in PDI in which the amino acid sequence of a and a' active sites, respectively, are written out in single-letter abbreviation.


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