Protein disulfide isomerase acts as a molecular chaperone during the assembly of procollagen.

Protein-disulfide isomerase (PDI) has been shown to be a multifunctional enzyme catalyzing the formation of disulfide bonds, as well as being a component of the enzymes prolyl 4-hydroxylase (P4-H) and microsomal triglyceride transfer protein. It has also been proposed to function as a molecular chaperone during the refolding of denatured proteins in vitro. To investigate the role of this multifunctional protein within a cellular context, we have established a semi-permeabilized cell system that reconstitutes the synthesis, folding, modification, and assembly of procollagen as they would occur in the cell. We demonstrate here that P4-H associates transiently with the triple helical domain during the assembly of procollagen. The release of P4-H from the triple helical domain coincides with assembly into a thermally stable triple helix. However, if triple helix formation is prevented, P4-H remains associated, suggesting a role for this enzyme in preventing aggregation of this domain. We also show that PDI associates independently with the C-propeptide of monomeric procollagen chains prior to trimer formation, indicating a role for this protein in coordinating the assembly of heterotrimeric molecules. This demonstrates that PDI has multiple functions in the folding of the same protein, that is, as a catalyst for disulfide bond formation, as a subunit of P4-H during proline hydroxylation, and independently as a molecular chaperone during chain assembly.