Intracellular degradation and reduced cell-surface expression of sucrase-isomaltase in heat-shocked Caco-2 cells.
To investigate the role of post-translational events in intestinal cell differentiation we have studied the effects of heat shock on processing and cell surface delivery of sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPPIV) and aminopeptidase N (APN) in Caco-2 cells. In cells cultured at 42.5 degrees C there was a rapid decline in sucrase activity, while DPPIV and APN were unaffected over a 3-day period. Immunofluorescence staining confirmed the selective disappearance of SI from the surface membrane after only 1 day of culture at 42.5 degrees C. Cell-surface biotinylation of cells metabolically labelled with [35S]methionine 4 h after a switch from 37 degrees C to 42.5 degrees C demonstrated that newly synthesized APN and DPPIV were associated with the surface membrane, while SI was almost completely retained intracellularly. Pulse-chase experiments confirmed that, in these cells, DPPIV and APN were normally processed and vectorially delivered to the cell surface; in contrast, conversion between the two conformationally distinct high-mannose precursor forms of SI (hmP1 and hmP2) was markedly inhibited, a significant fraction of newly synthesized enzyme was degraded, probably in the ER, and an immature form of complex-glycosylated SI precursor (cP) was produced and mostly retained intracellularly. Double labelling of Caco-2 cells for SI and cathepsin D excluded an accumulation of SI in the lysosomes, suggesting that this organelle was not involved in the degradation of SI. These results indicate that the ER may play an important role in intestinal cell differentiation by regulating the conformational maturation, degradation and eventual cellular localization of some digestive enzymes.
Quaroni
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A
Section of Physiology, Cornell University, Ithaca, NY 14853.
Paul
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EC
Nichols
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BL
D000230:Adenocarcinoma; D000626:Aminopeptidases / Q000378:metabolism; D000911:Antibodies, Monoclonal; D018826:CD13 Antigens; D002462:Cell Membrane / Q000201:enzymology; D003110:Colonic Neoplasms; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D008715:Methionine / Q000378:metabolism; D008871:Microvilli / Q000201:enzymology*; D011499:Protein Processing, Post-Translational; D013394:Sucrase-Isomaltase Complex / Q000096:biosynthesis / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D013462:Sulfur Radioisotopes; D008715:Methionine; D013394:Sucrase-Isomaltase Complex; D000626:Aminopeptidases; D018826:CD13 Antigens; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4