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 (approximately 90-110 kDa) than nonglycosylated NIS (approximately 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.
Levy
O
O
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
De la Vieja
A
A
Ginter
C S
CS
Riedel
C
C
Dai
G
G
Carrasco
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D006031:Glycosylation; D007454:Iodides / Q000378:metabolism; D017136:Ion Transport; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D027981:Symporters; D000099209:Sodium-Iodide Symporters
D002352:Carrier Proteins; D017931:DNA Primers; D007454:Iodides; D008565:Membrane Proteins; D027981:Symporters; D000099209:Sodium-Iodide Symporters