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.
Wilson
R
R
School of Biological Sciences, The University of Manchester, 2.205 Stopford Building, Manchester M13 9PT, United Kingdom.
Lees
J F
JF
Bulleid
N J
NJ
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003094:Collagen / Q000096:biosynthesis; D006900:Hydroxylation; D018832:Molecular Chaperones / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011347:Procollagen / Q000096:biosynthesis* / Q000737:chemistry; D014176:Protein Biosynthesis; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D018411:Spodoptera; D014158:Transcription, Genetic; D014162:Transfection
D018832:Molecular Chaperones; D011347:Procollagen; D011994:Recombinant Proteins; D003094:Collagen; D019704:Protein Disulfide-Isomerases