THE ROLE OF THE THIOL DISULFIDE CENTERS AND PEPTIDE BINDING-SITE IN THE CHAPERONE AND ANTI-CHAPERONE ACTIVITIES OF PROTEIN DISULFIDE-ISOMERASE/

The complexity of protein folding is often aggravated by the low solub ility of the denatured state. The inefficiency of the oxidative refold ing of reduced, denatured lysozyme results from a kinetic partitioning of the unfolded protein between pathways leading to aggregation and p athways leading to the native structure. Protein disulfide isomerase ( PDI), a resident foldase of the endoplasmic reticulum, catalyzes the i n vitro oxidative refolding of reduced, disulfide containing proteins, including denatured lysozyme. Depending on the concentrations of fold ase and denatured substrate and the order in which they are added to i nitiate folding, PDI can exhibit either a chaperone activity or an ant i-chaperone activity (Puig, A., and Gilbert, H. F. (1994) J. Biol. Che m 269, 7764-7771). PDI's chaperone activity leads to quantitative reco very of native lysozyme. Its anti-chaperone activity diverts substrate away from productive folding and facilitates disulfide cross-linking of lysozyme into large, inactive aggregates that specifically incorpor ate PDI. A mutant PDI (NmCm-PDI), in which both the N- and C-terminal active site cysteines have been changed to serines, loses all chaperon e activity and behaves as an anti chaperone at all substrate and PDI c oncentrations tested. The dithiol/disulfide sites of PDI are essential for the chaperone activity observed at high PDI concentrations, but t hey are not required for the anti-chaperone activity found at low PDI concentrations. Inactivation of PDI's peptide/protein binding site by a specific photoaffinity label (Noiva, R., Freedman, R. B., and Lennar z, W. J. (1993) J. Biol. Chem. 268, 19210-19217) inhibits the disulfid e isomerase and chaperone activity, but the protein still retains its anti-chaperone activity. In a glutathione redox buffer, lysozyme PDI a ggregates are disulfide crosslinked; however, disulfide cross-linking is not required for aggregate formation or for the incorporation of PD I into the aggregates. Although both the peptide binding site and the catalytic active sites of PDI are required for chaperone and disulfide isomerase activity, neither of these sites are involved in PDI's anti -chaperone activity. PDI's anti-chaperone activity could serve as a qu ality control device by providing an efficient mechanism to retain mis folded proteins in the endoplasmic reticulum (Marquardt, T., and Helen ius, A. (1992) J. Cell. Biol. 117, 505-513).