Protein disulphide isomerase (PDI) has been shown to be a multifunctional protein capable of catalysing disulphide-bond formation and isomerization, and of participating as a non-catalytic subunit of prolyl 4-hydroxylase (P4-H) and microsomal triacylglycerol transfer protein. It has also been proposed to function as a molecular chaperone during the refolding of denatured proteins in vitro. To investigate its potential role as a molecular chaperone within a cellular context, we studied the folding, modification and assembly of type X collagen in semi-permeabilized cells. Using this approach, we demonstrate that depletion of ATP has no effect on the rate or extent of helix formation, indicating that the individual triple helical regions do not interact with the molecular chaperone immunoglobulin heavy-chain binding protein (BiP). However, PDI was shown to interact transiently with type X during helix formation in a role related to its function as the β subunit of P4-H. Once the collagen triple helix was formed, PDI re-associated, indicating a role in preventing the premature assembly of this molecule into higher-order structures. This interaction was not thiol dependent, as a type X polypeptide that did not contain any cysteine residues was able to fold correctly and interact with PDI. Both PDI and the collagen-binding protein hsp47 showed a similar pH-dependent interaction with folded collagen, dissociating when the pH was lowered to pH 6.0. These results suggest a role for PDI in chaperoning type X collagen during its transport through the cell.

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