THIOL-INDEPENDENT INTERACTION OF PROTEIN DISULFIDE-ISOMERASE WITH TYPE-X COLLAGEN DURING INTRACELLULAR FOLDING AND ASSEMBLY

Protein disulphide isomerase (PDI) has been shown to be a multifunctio nal protein capable of catalysing disulphide-bond formation and isomer ization, and of participating as a noncatalytic subunit of prolyl 4-hy droxylase (P4-H) and microsomal triacylglycerol transfer protein. It h as 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 t he folding, modification and assembly of type X collagen in semipermea bilized cells. Using this approach, we demonstrate that depletion of A TP has no effect on the rate or extent of helix formation, indicating that the individual triple helical regions do not interact with the mo lecular chaperone immunoglobulin heavy-chain binding protein (BiP), Ho wever, PDI was shown to interact transiently with type X during helix formation in a role related to its function as the beta subunit of P4- H. Once the collagen triple helix was formed, PDI re-associated, indic ating a role in preventing the premature assembly of this molecule int o higher-order structures. This interaction was not thiol dependent, a s a type X polypeptide that did not contain any cysteine residues was able to fold correctly and interact with PDI. Both PDI and the collage n-binding protein hsp47 showed a similar pH-dependent interaction with folded collagen, dissociating when the pH was lowered to pH 6.0. Thes e results suggest a role for PDI in chaperoning type X collagen during its transport through the cell.