Role of Membrane-Bound Oxidoreductases in Protein Biogenesis
Research area: Protein Folding and Quality Control
Group leaders: Maurizio Molinari
Status: In progress
The lumen of the ER contains 23 PDI members that insure formation of the correct set of intra- and inter-molecular disulfide bonds as a crucial, rate-limiting reaction of the protein folding process (Figure 1A). The reason for this high redundancy of PDIs remains unclear. Certainly, individual members of the PDI family show tissue-specific distribution or some kind of substrate preference (e.g. ERp57 forms functional complexes with the ER lectins calnexin and calreticulin and acts upon their ligands). The aim of this project is to uncover the role in protein biogenesis of the 5 type I membrane-bound members of the PDI family (TMX1, TMX2, TMX3, TMX4 and TMX5) (Figure 1B). Active PDIs contain the characteristic CXXC active-site motif that engages folding substrates in so-called mixed disulfides (i.e. covalent bonds between a PDI and a substrate cysteine). Mixed disulfides are extremely short living intermediates of the protein folding reaction, which can be stabilized upon replacement of the second (resolving) cysteine residue in the PDIs catalytic site. These so-called PDIs “trapping mutants” have been used to capture endogenous substrates of select ER-resident oxidoreductases such as ERp57, PDI, P5, ERp18, ERp72, ERp46 and ERdj5. The expression of a TMX1 trapping mutant in the living cells and the characterization by mass spectrometry of the polypeptides remaining covalently bound to it revealed a selective association with a series of cysteine-containing membrane-bound proteins. This is in contrast to studies performed with trapping mutants of other PDIs, which were all found to associate both with soluble and membrane-bound endogenous substrates. Studies are ongoing to confirm the substrate topology-dependent specificity of TMX1 and to characterize the role in protein biogenesis of the other TMX proteins (Figure 2).
The PDI family. (A) The PDI family comprises 23 members. The 5 type I membrane-bound PDI family members (TMX proteins) are highlighted. (B) Domain structure of TMX1-TMX5.
PDIs interactors. Endogenous proteins associated with the ERdj5 and TMX1 trapping mutant are shown.