The nonactive site cysteine residues of yeast protein disulfide isomerase are not required for cell viability.
Protein disulfide isomerase (PDI), the product of the essential PDI1 gene of Saccharomyces cerevisiae catalyzes oxidization of thiols, reduction of disulfide bonds, and isomerization of disulfides. It can also act as a chaperone to facilitate folding of denatured proteins. The protein has 6 cysteine (Cys) residues. Four of these Cys are part of the 2 thioredoxin-like catalytic sites (-CGHC-), one of which is located near the N- and the other near the C-terminus. In addition, it has 2 non-active site Cys near the N-terminus. The function of these non-active site Cys of yeast PDI is poorly understood. Whereas in yeast PDI, these Cys residues are in the vicinity of the N-terminal-most active site, in mammalian PDI their position is closer to the C-terminal-most active site. We have examined their role and that of the active site cysteines by constructing an extensive set of mutants in which the Cys were systematically replaced by Ser. As reported earlier, the N-terminal Cys of the two active sites sequences of yeast PDI were found to be required for cell viability, but mutation of the C-terminal Cys to Ser in the two active sites was not lethal. We found that replacement of the two non-active site Cys with Ser did not affect cell viability, but in the case of the double mutant in which both Cys were replaced by Ser the processing and secretion of CPY was impaired.
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook 11794-5215, USA.
J M
JM
Luz
W J
WJ
Lennarz
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002110:Caffeine / Q000494:pharmacology; D003545:Cysteine; D004229:Dithiothreitol / Q000494:pharmacology; D008322:Mammals; D017354:Point Mutation; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development*; D012694:Serine
D002110:Caffeine; D012694:Serine; D019704:Protein Disulfide-Isomerases; D003545:Cysteine; D004229:Dithiothreitol