N-linked glycosylation of the thyroid Na⁺/I^- symporter (NIS). Implications for its secondary structure model

The Na⁺/I^- symporter (NIS), a 618-amino acid membrane glycoprotein that catalyzes the active accumulation of I^- into thyroid cells, was identified and characterized at the molecular level in our laboratory (Dai, G., Levy, O., and Carrasco, N. (1996) Nature 379, 458-460). Because mature NIS is highly glycosylated, it migrates in SDS-polyacrylamide gel electrophoresis as a broad polypeptide of higher molecular mass (~90-110 kDa) than nonglycosylated NIS (~50 kDa). Using site-directed mutagenesis, we substituted both separately and simultaneously the asparagine residues in all three putative N-linked glycosylation consensus sequences of NIS with glutamine and assessed the effects of the mutations on function and stability of NIS in COS cells. All mutants were active and displayed 50-90% of wild- type NIS activity, including the completely nonglycosylated triple mutant. This demonstrates that to a considerable extent, function and stability of NIS are preserved in the partial or even total absence of N-linked glycosylation. We also found that Asn²²⁵ is glycosylated, thus proving that the hydrophilic loop that contains this amino acid residue faces the extracellular milieu rather than the cytosol as previously suggested. We demonstrated that the NH₂ terminus faces extracellularly as well. A new secondary structure model consistent with these findings is proposed.