N-glycosylation controls functional activity of Oatp1, an organic anion transporter

Abstract

Rat Oatp1 (Slc21a1) is an organic anion-transporting polypeptide believed to be an anion exchanger. To characterize its mechanism of transport, Oatp1 was expressed in Saccharomyces cerevisiae under control of the GAL1 promoter. Protein was present at high levels in isolated S. cerevisiae secretory vesicles but had minimal posttranslational modifications and failed to exhibit taurocholate transport activity. Apparent molecular mass (M) of Oatp1 in yeast was similar to that of unmodified protein, approximately 62 kDa, whereas in liver plasma membranes Oatp1 has an M of approximately 85 kDa. To assess whether underglycosylation of Oatp1 in yeast suppressed functional activity, Oatp1 was expressed in Xenopus laevis oocytes with and without tunicamycin, a glycosylation inhibitor. With tunicamycin, M of Oatp1 decreased from approximately 72 to approximately 62 kDa and transport activity was nearly abolished. Mutations to four predicted N-glycosylation sites on Oatp1 (Asn to Asp at positions 62, 124, 135, and 492) revealed a cumulative effect on function of Oatp1, leading to total loss of taurocholate transport activity when all glycosylation sites were removed. M of the quadruple mutant was approximately 62 kDa, confirming that these asparagine residues are sites of glycosylation in Oatp1. Relatively little of the quadruple mutant was able to reach the plasma membrane, and most remained in unidentified intracellular compartments. In contrast, two of the triple mutants tested (N62/124/135D and N124/135/492D) were present in the plasma membrane fraction yet exhibited minimal transport activity. These results demonstrate that both membrane targeting and functional activity of Oatp1 are controlled by the extent of N-glycosylation.

Functional influence ofN-glycosylation in OCT2-mediated tetraethylammonium transport

“…Removal of glycosylation at two sites of OCT2 (N71/N96 and N71/N112) greatly diminished plasma membrane expression, and compared with the other mutants (single and double), plasma membrane expression of unglycosylated OCT2 was negligible. This observation is consistent with numerous other studies, in which elimination of glycosylation at multiple sites in a protein was observed to disrupt targeting to the plasma membrane (e.g., 15,29,39). In crude membranes, which consist of plasma membranes and membranes from intracellular sources, expression of the WT transporter and each of the mutants, except the triple mutant, was comparable.…”

“…Depending on both the species and cell type, the extent of glycosylation a protein receives may be different (14). For example, Kee et al (15) found that when expressed in yeast (N17 strain), the organic solute transporter, Oatp-1, is functionally inactive, an effect attributed to insufficient glycosylation. Here, we examined N-glycosylation of the rabbit ortholog of OCT2 using CHO cells.…”

Loss of N-Linked Glycosylation Reduces Urea Transporter UT-A1 Response to Vasopressin

“…In many cases, N-linked glycosylation is critical for membrane protein intracellular movement and its eventual delivery to the cell surface. Most membrane proteins targeted to the plasma membrane possess N-linked glycosylation (12,13,16,17,28). One type of hereditary AQP2 mutant (T125M), which causes recessive nephrogenic diabetes insipidus, is not glycosylated because the N-linked glycosylation motif is disrupted (29).…”

“…One type of hereditary AQP2 mutant (T125M), which causes recessive nephrogenic diabetes insipidus, is not glycosylated because the N-linked glycosylation motif is disrupted (29). Glycosylation has also been shown to be essential for the efficient function and surface expression of the NaCl cotransporter (14), the cardiac potassium channel HERG (30), and the Oatp1 organic anion transporter (16). We have previously shown that AVP and FSK can stimulate urea flux in MDCK cells stably expressing UT-A1 (24,25).…”

“…Inhibition of N-linked glycosylation can cause apoptosis (15). The effect of glycosylation on the functional activity of different types of membrane transport proteins has been described previously (7,16,17,(12)(13)(14). N-Glycosylation is required for functional activity of many membrane transporters; however, deglycosylation does not always abrogate transporter function (18,19).…”