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.

Regulation of Thyroid Oxidative State by Thioredoxin Reductase Has a Crucial Role in Thyroid Responses to Iodide Excess

“…Likewise, oxidation (in a reversible or irreversible way) of amino acids could generate an inactive protein. Moreover, there are indications that NIS may be a multimeric protein in its functional form (38,39). Redistribution or alteration of these multimeric forms by direct oxidation may also result in a rapid inactivation of NIS protein.…”

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

“…In mammalian cells, adding this extra sequence at the N -terminus of NIS protein has quite a negative effect, worse than the absence of a Kozak consensus sequence (data not shown). The addition of a Flag-tag epitope to the N -terminus of NIS also hampers its expression (yields, maturation) [93]. Because of these potential problems with protein expression, the constructs for expression in insect cells were designed not to contain the att sequence within the expressed protein.…”

Enhancing Expression of Functional Human Sodium Iodide Symporter and Somatostatin Receptor in Recombinant Oncolytic Vaccinia Virus for In Vivo Imaging of Tumors

“…Although specific hNIS expression was confirmed and as expected according to the literature by Western blot (Fig. 1D) (32,33,34), the functionality of the symporter was validated by measuring the accumulation of 131 I or 99m Tc radioisotopes into the cells (Figs. 1E and 1F).…”