title: Characterisation of the purified human sodium/iodide symporter reveals that the protein is mainly present in a dimeric form and permits the detailed study of a native C-terminal fragment.
abstract: The sodium/iodide symporter is an intrinsic membrane protein that actively transports iodide into thyroid follicular cells. It is a key element in thyroid hormone biosynthesis and in the radiotherapy of thyroid tumours and their metastases. Sodium/iodide symporter is a very hydrophobic protein that belongs to the family of sodium/solute symporters. As for many other membrane proteins, particularly mammalian ones, little is known about its biochemistry and structure. It is predicted to contain 13 transmembrane helices, with an N-terminus oriented extracellularly. The C-terminal, cytosolic domain contains approximately one hundred amino acid residues and bears most of the transporter's putative regulatory sites (phosphorylation, sumoylation, di-acide, di-leucine or PDZ-binding motifs). In this study, we report the establishment of eukaryotic cell lines stably expressing various human sodium/iodide symporter recombinant proteins, and the development of a purification protocol which allowed us to purify milligram quantities of the human transporter. The quaternary structure of membrane transporters is considered to be essential for their function and regulation. Here, the oligomeric state of human sodium/iodide symporter was analysed for the first time using purified protein, by size exclusion chromatography and light scattering spectroscopy, revealing that the protein exists mainly as a dimer which is stabilised by a disulfide bridge. In addition, the existence of a sodium/iodide symporter C-terminal fragment interacting with the protein was also highlighted. We have shown that this fragment exists in various species and cell types, and demonstrated that it contains the amino-acids [512-643] from the human sodium/iodide symporter protein and, therefore, the last predicted transmembrane helix. Expression of either the [1-512] truncated domain or the [512-643] domain alone, as well as co-expression of the two fragments, was performed, and revealed that co-expression of [1-512] with [512-643] allowed the reconstitution of a functional protein. These findings constitute an important step towards an understanding of some of the post-translational mechanisms that finely tune iodide accumulation through human sodium/iodide symporter regulation.
authors.affiliation: CEA, iBEB, SBTN, Centre de Marcoule, Bat 170, BP17171, 30207 Bagnols sur Cèze, CEDEX, France. sylvaine.huc@igf.cnrs.fr
authors.forename: Sylvaine
authors.initials: S
authors.lastname: Huc-Brandt
authors.forename: Didier
authors.initials: D
authors.lastname: Marcellin
authors.forename: Fanny
authors.initials: F
authors.lastname: Graslin
authors.forename: Olivier
authors.initials: O
authors.lastname: Averseng
authors.forename: Laurent
authors.initials: L
authors.lastname: Bellanger
authors.forename: Patrick
authors.initials: P
authors.lastname: Hivin
authors.forename: Eric
authors.initials: E
authors.lastname: Quemeneur
authors.forename: Cécile
authors.initials: C
authors.lastname: Basquin
authors.forename: Valérie
authors.initials: V
authors.lastname: Navarro
authors.forename: Thierry
authors.initials: T
authors.lastname: Pourcher
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Darrouzet
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D001671:Biochemistry / Q000379:methods; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012974:Sodium Iodide / Q000737:chemistry; D027981:Symporters / Q000737:chemistry*; D013961:Thyroid Gland / Q000378:metabolism; D000099209:Sodium-Iodide Symporters
chemical_list: D000596:Amino Acids; D004220:Disulfides; D011994:Recombinant Proteins; D012974:Sodium Iodide; D027981:Symporters; D000099209:Sodium-Iodide Symporters