title: PDI-mediated ER retention and proteasomal degradation of procollagen I in corneal endothelial cells.
abstract: Procollagen I in corneal endothelial cells (CECs) is intracellularly degraded immediately after its synthesis. In this study, we investigated the mechanism of intracellular degradation of procollagen I by determining the role of protein disulfide isomerase (PDI) in endoplasmic reticulum (ER) retention and further determined the degradation pathway of procollagen I in CECs. When association of PDI to monomeric proalpha chains or the trimeric procollagen I carboxyl propeptides (PICPs) was analyzed, immune complex precipitated with anti-PICP antibody contained more PDI than that precipitated with antibodies to monomeric chains. PICPs were completely colocalized with PDI. When CECs were transfected with PDI vector, procollagen I and the recombinant PDI were colocalized in the ER, whereas CECs transfected with PDI minus KDEL (the ER retrieval sequence) vector demonstrated that the two proteins were localized in the Golgi and were subsequently secreted into the medium. Ribostamycin (an inhibitor of the chaperone activity of PDI) blocked colocalization of PDI and procollagen I. Cells treated with chloroquine (lysosome inhibitor) did not alter the subcellular localization of procollagen I, because the inhibitor failed to induce the accumulation of procollagen I at Golgi. On the other hand, procollagen I was colocalized with ubiquitin in the cytoplasm, and proteasomal inhibitors further facilitated the colocalization of the two proteins and accumulation of ubiquitinated procollagen I ladders. These results suggest that association of PDI with procollagen I, whether monomeric or trimeric, leads to ER retention of procollagen I before intracellular degradation via the ubiquitin-proteasome pathway.
authors.affiliation: Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90089-9092, USA.
authors.forename: MinHee K
authors.initials: MK
authors.lastname: Ko
authors.forename: EunDuck P
authors.initials: EP
authors.lastname: Kay
mesh_terms: D000818:Animals; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000502:physiology*; D019573:Epithelium, Corneal / Q000166:cytology / Q000502:physiology*; D015151:Immunoblotting; D009097:Multienzyme Complexes / Q000378:metabolism*; D011347:Procollagen / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
chemical_list: D009097:Multienzyme Complexes; D011347:Procollagen; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D019704:Protein Disulfide-Isomerases