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 cel l differentiation we have studied the effects of heat shock on process ing and cell surface delivery of sucrase-isomaltase (SI), dipeptidylpe ptidase IV (DPPIV) and aminopeptidase N (APN) in Caco-2 cells. In cell s cultured at 42.5-degrees-C there was a rapid decline in sucrase acti vity, while DPPIV and APN were unaffected over a 3-day period. Immunof luorescence staining confirmed the selective disappearance of SI from the surface membrane after only 1 day of culture at 42.5-degrees-C. Ce ll-surface biotinylation of cells metabolically labelled with [S-35]me thionine 4 h after a switch from 37-degrees-C to 42.5-degrees-C demons trated that newly synthesized APN and DPPIV were associated with the s urface membrane, while SI was almost completely retained intracellular ly. Pulse-chase experiments confirmed that, in these cells, DPPIV and APN were normally processed and vectorially delivered to the cell surf ace; in contrast, conversion between the two conformationally distinct high-mannose precursor forms of SI (hmp1 and hmP2) was markedly inhib ited, a significant fraction of newly synthesized enzyme was degraded, probably in the ER, and an immature form of complex-glycosylated SI p recursor (cP) was produced and mostly retained intracellularly. Double labelling of Caco-2 cells for SI and cathepsin D excluded an accumula tion of SI in the lysosomes, suggesting that this organelle was not in volved in the degradation of SI. These results indicate that the ER ma y play an important role in intestinal cell differentiation by regulat ing the conformational maturation, degradation and eventual cellular l ocalization of some digestive enzymes.