Role of Ero1 and PDI in oxidative folding

Perhaps the most distinctive feature of protein folding in the ER is the abundance of disulfide bonds that must form during maturation of proteins traveling along the secretory pathway. Formation of disulfide bonds is a redox reaction. Thus, to match the flux of disulfide bonds that exit from the ER by virtue of protein secretion, a flux of oxidizing equivalents into the ER is required. In eukaryotic cells, the essential protein relay supporting this flux, and hence disulfide bond formation, involves Ero1 and protein disulfide isomerase (PDI). Client proteins are oxidized by PDI, which renders PDI in a reduced free sulfhydryl containing state. Ero1 recharges PDI, whereby its cofactor flavin adenine dinucleotide (FAD) is reduced to FADH₂. Ero1 then uses molecular oxygen as terminal electron acceptor, converting FADH₂ into FAD and H₂O.

PDI contains two CXXC motifs, which form the active sites of the oxidoreductase. When PDI is oxidized (i.e. the two cysteines in the active site are disulfide bonded), PDI can function as an oxidase for client proteins that emerge in the ER lumen. The active site disulfides are unstable and easily disrupted by accessible free sulfhydryl groups in the nascent client protein, yielding a mixed disulfide between PDI and the client protein instead. A second free sulfhydryl of the nascent chain can subsequently disrupt the mixed disulfide, resulting in a disulfide between two substrate protein cysteines. As a consequence PDI is released in reduced form. Reduced PDI in turn can catalyze isomerization of disulfides. The N-terminal free cysteine in the active site of reduced PDI can disrupt a pre-existing disulfide bond of a substrate protein, resulting in a mixed disulfide between substrate and PDI, similarly as in the oxidase reaction of PDI. The disulfide isomerization cycle is complete when this cysteine teams up with another cysteine than its former partner.

Typically, Ero1 variants contain a CXXCXXC motif. We have shown that the intact CXXCXXC motif is required for the folding, stability and structure of human Ero1alpha. The CXXCXXC motif is also essential for covalent interactions of Ero1 with PDI, while covalent interactions with PDI-related proteins, Erp57, Erp72 and P5 could not be detected. Expression levels of Ero1-alpha and -beta influence the rate and efficiency of disulfide bond formation of the antibody subunits J-chain and kappa-chain. Mutants of the CXXCXXC domain can act as dominant negatives for oxidative folding.

Mezghrani A, Fassio A, Benham A, Simmen T, Braakman I & Sitia R Manipulation of oxidative protein folding and PDI redox state in mammalian cells. EMBO J. 2001; 15:6288-96. pdf
Benham AM, Cabibbo A, Fassio A, Bulleid N, Sitia R & Braakman I The CXXCXXC motif determines the folding, structure and stability of human Ero1-Lalpha. EMBO J. 2000;19:4493-502. pdf

Currently, we investigate the influence of Ero1 on the oxidative folding of model proteins LDL receptor, HIV Envelope, Influenza HA and CFTR. In addition, we study the interactions of Ero1 with PDI and with a novel PDI-related ER resident protein, ERp44.

• The Ero1 crew
  Ineke Braakman
  Jenny Smit (alumna)
  Adam Benham (alumnus)