The secretory pathway is normal in dithiothreitol-treated cells, but disulfide-bonded proteins are reduced and reversibly retained in the endoplasmic reticulum.
Addition of 2 mM dithiothreitol (DTT) to HepG2 human hepatoma cells blocks secretion of newly made albumin and causes it to accumulate in the endoplasmic reticulum in the reduced state. Subsequent incubation of the cells in the absence of DTT allows the reduced albumin to form apparently normal disulfide bonds and to be secreted normally. Similarly, DTT treatment causes all newly made subunits of the H1 subunit of the asialoglycoprotein receptor to be retained in the endoplasmic reticulum in the reduced state; following removal of DTT H1 subunits form disulfide bonds via a normal endoplasmic reticulum folding intermediate and mature to the Golgi. Thus, apparently normal formation of disulfide bonds on these two proteins can occur post-translationally, as has been shown previously for the influenza HA hemagglutinin (Braakman, I., Helenius, J., and Helenius, A. (1992) EMBO J. 11, 1717-1722; Braakman, I., Helenius, J., and Helenius, A. (1992) Nature 356, 260-262). alpha 1-Antitrypsin contains no disulfide bonds; in the continuous presence of DTT it acquires a normal complement of complex oligosaccharides and is secreted at an only slightly reduced rate. Thus, the secretory pathway functions efficiently even when it is reduced by DTT. Endoplasmic reticulum retention of albumin and H1 during treatment with DTT presumably occurs because, without disulfide bonds, these proteins cannot fold properly, not because of any defect in the overall processes of vesicular transport and protein secretion.
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142.
H F
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Lodish
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Kong
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism; D001345:Autoradiography; D000001:Calcimycin / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular; D002499:Centrifugation, Density Gradient; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D011971:Receptors, Immunologic / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012260:Ribonucleases / Q000494:pharmacology; D012709:Serum Albumin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014168:Transferrin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D004220:Disulfides; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D011971:Receptors, Immunologic; D012709:Serum Albumin; D013462:Sulfur Radioisotopes; D014168:Transferrin; D000515:alpha 1-Antitrypsin; D000001:Calcimycin; D008715:Methionine; D012260:Ribonucleases; D003545:Cysteine; D004229:Dithiothreitol