Functional expression and characterization of the wild-type mammalian renal cortex sodium/phosphate cotransporter and an R-215 mutant in Saccharomyces cerevisiae

The wild-type and an R215E mutant of the rat renal cortex sodium/phosphate cotransporter type 2 (NaPi-2) were functionally expressed in the yeast Sacc haromyces cerevisiae strain MB192, a cell line lacking the high-affinity en dogenous H+/Pi cotransporter. The expression of the mRNA molecules and corr esponding proteins was confirmed by Northern and Western blot analysis, res pectively. As detected by indirect immunofluorescence and antibody capture assay, both wild-type and mutant NaPi-2 proteins are expressed in the yeast plasma membrane in comparable amounts. In the presence of 5 mu M phosphate , Na+ promotes phosphate uptake into yeast cells expressing the wild-type N aPi-2 with a K-0.5 of 5.6 +/- 1.1 mM. The maximum uptake of phosphate (649 +/- 30 pmol/10 min) is approximately 8-fold higher than the uptake obtained with nontransformed cells (76.8 +/- 8 pmol/10 min). Yeast cells expressing the R215E mutant of NaPi-2 accumulate 213 +/- 9 pmol of phosphate/10 min u nder the same conditions. The K0.5 for the stimulation of phosphate uptake by Nai is 4.2 +/- 0.8 mM for the R215E mutant and thus not significantly di fferent from the value obtained with cells expressing the wild-type cotrans porter. The reduced level of accumulation of phosphate in yeast cells expre ssing the R215E mutant is probably due to a reduction of the first-order ra te constant k for phosphate uptake: while cells expressing wild-type NaPi-2 accumulate phosphate with a k of 0.06 min(-1), the rate for phosphate upta ke into cells expressing the R215E mutant (k) is 0.016 min(-1) and therefor e about 4-fold lower. In comparison, the rate for phosphate uptake into non transformed cells (k) is 0.0075 min(-1). Phosphate uptake into yeast cells that express the wild-type NaPi-2 in the presence of 150 mM NaCl is promote d by extracellular phosphate with a K0.5 of 45 +/- 4 mu M. A phosphate-depe ndent phosphate accumulation is also observed with cells expressing the R21 5E mutant, but the K0.5 is twice as high (86 +/- 5 mu M) as that obtained w ith the wild-type cotransporter. We conclude that the yeast expression syst em is a useful tool for the investigation of structure-function relationshi ps of the renal sodium/phosphate cotransporter and that R-215, although not involved in Na+ recognition, is a part of the structure involved in phosph ate recognition and considerably influences the rate of phosphate uptake by the NaPi-2 cotransporter.