A. Quaroni et al., INTRACELLULAR DEGRADATION AND REDUCED CELL-SURFACE EXPRESSION OF SUCRASE-ISOMALTASE IN HEAT-SHOCKED CACO-2 CELLS, Biochemical journal, 292, 1993, pp. 725-734
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.