Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum.
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.
Holst
B
B
Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark.
Tachibana
C
C
Winther
J R
JR
D001665:Binding Sites / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology*; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D006031:Glycosylation; D007535:Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis / Q000502:physiology; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D013439:Sulfhydryl Reagents; D013879:Thioredoxins; D007535:Isomerases; C076879:EUG1 protein, S cerevisiae; D019704:Protein Disulfide-Isomerases; D004229:Dithiothreitol