Abstract
Recently we found by immunogold electron microscopy that protein disulfide-isomerase (PDI), a major resident protein in the lumen of the endoplasmic reticulum (ER) of many cells, is exceptionally localized in rat exocrine pancreatic cells not only in the ER but also in plasma membranes and other organelles along secretory pathway (Akagi, S., Yamamoto, A., Yoshimori, T., Masaki, R., Ogawa, R., and Tashiro, Y. (1988) J. Histochem. Cytochem. 36, 1069-1074). These observations suggest that another type of PDI, e.g. one with a defective ER retention signal, might exist and be transported in the exocrine pancreatic cells. We therefore compared biochemical and immunochemical properties of the transported PDI with the authentic ER resident PDI. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, urea-polyacrylamide gel electrophoresis, and isoelectric focusing showed that the former was indistinguishable from the latter. We prepared a polyclonal antibody against the synthetic hexapeptide, which corresponds to the carboxyl terminus of PDI containing the putative ER retention signal "KDEL." The epitopes of this antibody (anti-KDEL antibody) were located within the KDEL sequence. Anti-KDEL antibody reacted with PDI in both the plasma membranes and the ER of rat pancreatic cells in immunoblot analysis as well as in immunogold electron microscopy. These results suggest that PDI exported from the ER to the plasma membranes in rat exocrine pancreatic cells possesses the KDEL sequence.
Highlights
Protein Disulfide-isomerase in Rat Exocrine Pancreatic Cells Is Exported from the Endoplasmic Reticulum Despite Possessing the Retention Signal*
We found by immunogold electron microscopy that protein disulfide-isomerase (PDI), a major resident protein in the lumen of the endoplasmic reticulum (ER) of many cells, is exceptionally localized in rat exocrine pancreatic cells in the ER and in plasma membranes and other organelles along secretory pathway
Protein disulfide-isomerase (PDI) in both the plasma membranes and the ER of rat pancreatic cells in immunoblot analysis as well as in immunogold electron microscopy. These results suggest that PDI exported from the ER to the plasma membranes in rat exocrine pancreatic cells possesses the KDEL sequence
Summary
Materials-The following materials were used: [%]methionine (ICN Radiochemicals, Irvine, CA); autoradiography enhancer. Peptide Map_p_ing--Secreted and cellular PDI and the cross-reacting protein in ARIP cells were subjected to partial proteolytic digestion with S. aureus nrotease and subsequent SDS-PAGE as described previously (Stein and Sussman, 1983).-. Immunoblot Analysis-Gels of SDS-PAGE, urea-PAGE, and isoelectric focusing were immunoblotted with anti-PDI antibody or affinity-purified anti-KDEL antibody and peroxidase-conjugated anti-rabbit IgG essentially according to the method of Burnette (1981). Binding Assay-Binding activity of anti-KDEL antibody to the peptide AVKDEL and GTGEEDTSEKDEL was assayed by ELISA according to the method described elsewhere (Yoshimori et al, 1988). Zmmunogold Electron Microscopy-The ultrastructural localization of binding sites of anti-KDEL antibody in rat exocrine pancreatic cells was investigated by the postembedding protein A-gold technique as described elsewhere (Akagi et al, 198813). For fixation of the pancreas, rats were perfused with 4% formaldehyde in 0.1 M cacodylate buffer (pH 7.4). 60 pg/ml anti-KDEL antibody or rabbit IgG from preimmune rabbits was used for control
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