Intracellular traffic of the ecto-nucleotide pyrophosphatase/phosphodiesterase NPP3 to the epical plasma membrane of MDCK and Caco-2 cells: apical targeting occurs in the absence of N-glycosylation

Glycosylation was considered the major signal candidate for apical targetin g of transmembrane proteins in polarized epithelial cells. However, direct demonstration of the role of glycosylation has proved difficult because non glycosylated apical transmembrane proteins usually do not reach the cell su rface. Here we were able to follow the targeting of the epical transmembran e glycoprotein NPP3 both when glycosylated and non-glycosylated, Transfecte d in polarized MDCK and Caco-2 cells, NPP3 was exclusively expressed at the apical membrane. The transport kinetics of the protein to the cell surface were studied after metabolic S-35-labeling and surface immunoprecipitation , The newly synthesized protein was mainly targeted directly to the apical surface in MDCK cells, whereas 50% transited through the basolateral surfac e in Caco-2 cells, In both cell types, the basolaterally targeted pool was effectively transcytosed to the apical surface, In the presence of tunicamy cin, NPP3 was not N-glycosylated. The nonglycosylated protein was partially retained intracellularly but the fraction that reached the cell surface wa s nevertheless predominantly targeted apically, However, transcytosis of th e non-glycosylated protein was partially impaired in MDCK cells, These resu lts provide direct evidence that glycosylation cannot be considered an apic al targeting signal for NPP3, although glycosylation is necessary for corre ct trafficking of the protein to the cell surface.