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 (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 [(35)S]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 degrees 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.
Wick
D A
DA
Department of Biochemistry, Division of Gastroenterology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Seetharam
B
B
Dahms
N M
NM
D018938:Caco-2 Cells; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D007425:Intracellular Membranes / Q000378:metabolism; D008024:Ligands; D017527:Receptor, IGF Type 2 / Q000378:metabolism*
D008024:Ligands; D017527:Receptor, IGF Type 2; D007335:Insulin-Like Growth Factor II