The role of N-linked glycosylation in protein folding, membrane targeting, and substrate binding of human organic anion transporter hOAT4

We used a novel approach to evaluate how the addition/acqui-sition and processing/modification of N-linked oligosaccha-rides play a role in the functional maturation of human organic anion transporter hOAT4. Inhibition of acquisition of oligosac-charides in hOAT4 by mutating asparagine to glutamine and by tunicamycin treatment was combined with the expression of wild-type hOAT4 in a series of mutant Chinese hamster ovary (CHO)-Lec cells defective in the different steps of glycosylation processing. We showed that both the disruption of the glyco-sylation sites by mutagenesis and the inhibition of glycosylation by tunicamycin treatment resulted in a nonglycosylated hOAT4, which was unable to target to the cell surface. In contrast, hOAT4 synthesized in mutant CHO-Lec cells, carrying different structural forms of sugar moieties (mannose-rich in Lec1 cells