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* Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark; and Aspects of protein disulfide isomerase (PDI)
function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a Department of Biochemistry and Molecular Biology, Penn State University, University Park, Pennsylvania 16802
, 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 SGHS. 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.
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