Cysteine residues and the structure of the rat renal proximal tubular typeII sodium phosphate cotransporter (Rat NaPi IIa)

The rat renal Na/P-i cotransporter type IIa (rat NaPi IIa) is a 637 amino a cid protein containing 12 cysteine residues, We examined the effect of diff erent cysteine modifying methanethiosulfonate (MTS)-reagents and the disulf ide bond reducing agent tris(2-carboxyethyl)phosphine (TCEP) on the transpo rt activity of wild-type and 12 single cysteine substitution mutants of mt NaPi IIa expressed in Xenopus laevis oocytes. The transport activity of the wild-type protein was resistant to three membrane impermeant MTS-reagents (MTSEA, MTSET and MTSES). In contrast, membrane permeant methyl methanethio sulfonate (MMTS) and TCEP inhibited the transport activity of both the wild -type, as well as all the single mutant proteins. This indicated the existe nce of mole than one functionally important cysteine residue, not accessibl e extracellularly, and at least 2 disulfide bridges. To identify the disulf ide bridges, three double mutants lacking 2 of the 3 cysteine residues pred icted to be extracellular in different combinations were examined. This led to the identification of one disulfide bridge between C306 and C334; recon sideration of the topological model predictions suggested a second disulfid e bridge between C225 and C520. Evaluation of a fourth double mutant indica ted that at least one of two disulfide bridges (C306 and C334; C225 and C52 0) has to be formed to allow the surface expression of a functional cotrans porter. A revised secondary structure is proposed which includes two partia lly repeated motifs that are connected by disulfide bridges formed between cysteine pairs C306-C334 and C225-C520.