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MK Ko, EP Kay; Role of Protein Disulfide Isomerase for Retention of Procollagen I in Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3188.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Corneal endothelial cells (CEC) synthesize procollagen I and degrade it intracellularly. We have shown that protein disulfide isomerase (PDI) plays a key role in procollagen I retention in the endoplasmic reticulum (ER). In this study, we investigated whether PDI associates with procollagen I via the individual C-propeptide chains or via the trimeric C-propeptide. We further studied whether the KDEL sequence was important for ER retention of procollagen I. Methods: Protein colocalization was determined by double staining. Protein-protein interaction was determined by coimmunoprecipitation of the cross-linked cell lysate or medium sample followed by immunoblotting. CEC were transfected for 24 h with PDI minus KDEL (PDI-KDEL) vector or with PDI vector. Results: To determine whether PDI interacted with monomeric procollagen I chains or with the trimeric molecule, the cross-linked cell lysate was first precipitated with anti-proα1(I), proα2(I), procollagen I carboxyl-terminal propeptide (PICP), or type I collagen antibody. The lysate was then immunoblotted with PDI antibody. The immune complex precipitated with anti-PICP antibody contained far more PDI than those precipitated with the other antibodies. PICP largely colocalized with PDI. To investigate whether KDEL sequence was essential for ER retention, medium fraction obtained from CEC transfected with PDI-KDEL vector was immunoprecipitated with anti-PICP antibody and immunoblotted with anti-HA antibody (tagged protein). CEC transfected with PDI-KDEL secreted the protein complex, whereas cells transfected with PDI vector did not secrete PDI. Colocalization of PDI-KDEL with PDI demonstrated that PDI showed an ER distribution, while PDI-KDEL molecule showed a perinuclear Golgi profile. Conclusion: These data suggest that PDI associates with procollagen I via the trimeric carboxy-terminal propeptide and that the KDEL sequence of PDI is essential for ER retention of both the cargo protein and the enzyme.
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