AGI September 40/3

Wick, Debra A., Bellur Seetharam, and Nancy M. Dahms. Biosynthesis and secretion of the mannose 6-phosphate receptor and its ligands in polarized Caco-2 cells. Am. J. Physiol. 277 (Gastrointest. Liver Physiol. 40): G506–G514, 1999.—We have analyzed the transport of newly synthesized mannose 6-phosphate (Man-6-P)-bearing proteins (i.e., lysosomal enzymes) in the polarized human colon adenocarcinoma cell line, Caco-2, by subjecting filter-grown cells to a pulse-chase labeling protocol using [35S]methionine, and the resulting cell lysate, apical medium, and basolateral medium were immunoprecipitated with insulin-like growth factor II/Man-6-P receptor (IGF-II/MPR)-specific antisera. The results showed that the majority of secreted lysosomal enzymes accumulated in the apical medium at .2 h of chase and that this polarized distribution was facilitated by the IGF-II/MPR selectively endocytosing lysosomal enzymes from the basolateral surface. Treatment with various agents known to affect vesicular transport events demonstrated that incubations at 16°C or incubations with brefeldin A inhibited the secretion of lysosomal enzymes from both the apical and basolateral surface, whereas treatment with nocodazole selectively blocked apical secretion. In contrast, incubation with NH4Cl or nocodazole had a stimulatory effect on basolateral secretion. Taken together, these results demonstrate that the sorting of Man-6-P-containing proteins into the apical and basolateral secretory pathways is regulated by distinct components of the intracellular trafficking machinery.