One of the rate-limiting steps in the folding pathways of many secretory proteins is the formation of correct disulfide bonds between cysteine residues. In eukaryotes, both disulfide bond formation and isomerisation which shuffles incorrectly formed disulfides are catalysed by protein disulfide isomerase (PDI), whereas in bacteria these two reactions are catalysed by separate enzymes. Both in eukaryotic and prokaryotic cells the oxidation and isomerisation steps proceed exclusively in extracytoplasmic environments (the lumen of the eukaryotic endoplasmic reticulum and the Gram-negative bacterial periplasmic space). The family of foldases under discussion is characterised by a conserved thioredoxin fold and a common active site motif: Cys-X-X-Cys. The process of disulfide bond formation relies on thiol-disulfide exchange between oxidised and reduced cysteine pairs in the catalyst and substrate protein. Two separate pathways involved in disulfide bond formation and isomerisation have been characterised both in eukaryotes and in bacteria. In the oxidative pathway, oxidizing equivalents flow from oxygen to a membrane protein (Ero1p in eukaryotes or DsbB in bacteria), and then to a folding protein containing reduced cysteines via PDI (in eukaryotes) or via DsbA (in bacteria). In the isomerisation pathway, DsbC (bacterial protein disulfide isomerase) or PDI (in eukaryotes) interacts with substrate proteins that contain non-native disulfide bonds, allowing these bonds to rearrange to their native pairings. Reducing equivalents which are necessary to maintain DsbC in a reduced form, able to attack misfolded disulfides, are transferred from the cytoplasm with the aid of the cytoplasmic membrane protein DsbD. In eukaryotes, reduced glutathione is the main source of reducing equivalents for PDI. A dual role of PDI as an oxidase and an isomerase is facilitated by its complex domain architecture.
