Biosynthesis and secretion of the mannose 6-phosphate receptor and its ligands in polarized Caco-2 cells

We have analyzed the transport of newly synthesized mannose 6-phosphate (Ma n-6-P)-bearing proteins (i.e., lysosomal enzymes) in the polarized human co lon adenocarcinoma cell line, Caco-2, by subjecting filter-grown cells to a pulse-chase labeling protocol using [S-35]methionine, and the resulting ce ll lysate, apical medium, and basolateral medium were immunoprecipitated wi th insulin-like growth factor II/Man-6-P receptor (IGF-II/MPR)-specific ant isera. The results showed that the majority of secreted lysosomal enzymes a ccumulated in the apical medium at >2 h of chase and that this polarized di stribution was facilitated by the IGF-II/MPR selectively endocytosing lysos omal enzymes from the basolateral surface. Treatment with various agents kn own to affect vesicular transport events demonstrated that incubations at 1 6 degrees C or incubations with brefeldin A inhibited the secretion of lyso somal enzymes from both the apical and basolateral surface, whereas treatme nt with nocodazole selectively blocked apical secretion. In contrast, incub ation with NH4Cl or nocodazole had a stimulatory effect on basolateral secr etion. Taken together, these results demonstrate that the sorting of Man-B- P-containing proteins into the apical and basolateral secretory pathways is regulated by distinct components of the intracellular trafficking machiner y.