Dithiothreitol treatment of Madin-Darby canine kidney cells reversibly blocks export from the endoplasmic reticulum but does not affect vectorial targeting of secretory proteins.
Addition of dithiothreitol (DTT) to the culture medium of Madin-Darby canine kidney (MDCK) cells blocks transport of newly synthesized gp80 (clusterin, apolipoprotein J), a soluble marker protein for apical exocytosis in this epithelial cell line. In cells treated with DTT during pulse labeling, gp80 is retained in the endoplasmic reticulum. After removal of the reducing agent, gp80 is posttranslationally oxidized and secreted at the apical surface of MDCK cell monolayers. This demonstrates that when folded and oxidized posttranslationally, gp80 can acquire a conformation that exhibits sorting signals for vectorial targeting. In the continuous presence of DTT, the transepithelial electrical resistance of filter-grown monolayers is maintained for several hours, indicating the presence of functionally intact tight junctions. Under the same conditions, transport competent forms of gp80 mature within the Golgi complex and are secreted predominantly at the apical surface of MDCK monolayers. Furthermore, another secretory protein, the osteopontin-derived 20-kDa polypeptide, is targeted to the apical plasma membrane domain in the continuous presence of DTT. These results suggest that the apical sorting machinery in MDCK cells functions independent of the redox state of the secretory pathway.
Lösch
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Institut für Biochemie, Johannes Gutenberg-Universität, Mainz, Germany.
Koch-Brandt
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D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D051152:Clusterin; D003545:Cysteine / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology*; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D004848:Epithelium / Q000187:drug effects / Q000502:physiology; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D007668:Kidney; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008715:Methionine / Q000378:metabolism; D018832:Molecular Chaperones; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D012471:Salivary Proteins and Peptides / Q000096:biosynthesis; D013462:Sulfur Radioisotopes
D051152:Clusterin; D006023:Glycoproteins; D018832:Molecular Chaperones; D012471:Salivary Proteins and Peptides; D013462:Sulfur Radioisotopes; D008715:Methionine; D003545:Cysteine; D004229:Dithiothreitol