Abstract
The formation of disulfides in proteins entering the secretory pathway is catalysed by the protein disulfide isomerase (PDI) family of enzymes. These enzymes catalyse the introduction, reduction and isomerization of disulfides. To function continuously they require an oxidase to reform the disulfide at their active site. To determine how each family member can be recycled to catalyse disulfide exchange, we have studied whether disulfides are transferred between individual PDI family members. We studied disulfide exchange either between purified proteins or by identifying mixed disulfide formation within cells grown in culture. We show that disulfide exchange occurs efficiently and reversibly between specific PDIs. These results have allowed us to define a hierarchy for members of the PDI family, in terms of ability to act as electron acceptors or donors during thiol-disulfide exchange reactions and indicate that there is no kinetic barrier to the exchange of disulfides between several PDI proteins. Such promiscuous disulfide exchange negates the necessity for each enzyme to be oxidized by Ero1 (ER oxidoreductin 1) or reduced by a reductive system. The lack of kinetic separation of the oxidative and reductive pathways in mammalian cells contrasts sharply with the equivalent systems for native disulfide formation within the bacterial periplasm.
Highlights
A family of proteins, collectively termed the protein disulfide isomerases (PDIs), is responsible for formation of correct disulfides in proteins entering the secretory pathway
In an attempt to extend our understanding of how individual PDI family members are oxidized or reduced, we have examined the ability of various PDI proteins to exchange disulfides with one another
Disulfide bond formation catalysed by the PDI proteins typically involves a bimolecular nucleophilic substitution reaction (SN2) initiated by the attack on a disulfide bond by a nucleophilic thiolate anion
Summary
A family of proteins, collectively termed the protein disulfide isomerases (PDIs), is responsible for formation of correct disulfides in proteins entering the secretory pathway. PDI proteins typically catalyse thioldisulfide exchange reactions Whether these enzymes function in the formation or breaking of a disulfide depends on the reduction potential of their CXXC active site disulfide, their ability to be recycled efficiently and their functional interaction with their substrate proteins [5,6]. The necessity to reduce disulfides, both non-native and native, by PDI proteins is crucial during folding of complex disulfide-containing polypeptides, during ER-associated degradation (ERAD) and in the regulation of the unfolded protein response (UPR) [18,19,20] The consequence of this reduction is that the PDI proteins will be oxidized requiring a reductive pathway to recycle the enzyme for further activity.
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