Identification of conserved prolyl residue important for transport activity and the substrate specificity range of yeast plasma membrane Na+/H+ antiporters

Yeast plasma membrane Na+/H+ antiporters are divided according to their substrate specificity in two distinct subfamilies. To identify amino acid residues responsible for substrate specificity determination ( recognition of K+), the Zygosaccharomyces rouxii Sod2-22 antiporter ( non-transporting K+) was mutagenized and a collection of ZrSod2-22 mutants that improved the KCl tolerance of a salt-sensitive Saccharomyces cerevisiae strain was isolated. Several independent ZrSod2-22 mutated alleles contained the replacement of a highly conserved proline 145 with a residue containing a hydroxyl group ( Ser, Thr). Site-directed mutagenesis of Pro(145) proved that an amino acid with a hydroxyl group at this position is enough to enable ZrSod2-22p to transport K+. Simultaneously, the P145( S/T) mutation decreased the antiporter transport activity for both Na+ and Li+. Replacement of Pro(145) with glycine resulted in a ZrSod2-22p with extremely low activity only for Na+, and the exchange of a charged residue ( Asp, Lys) for Pro(145) completely stopped the activity. Mutagenesis of the corresponding proline in the S. cerevisiae Nha1 antiporter ( Pro(146)) confirmed that this proline of the fifth transmembrane domain is a critical residue for antiporter function. This is the first evidence that a non-polar amino acid residue is important for the substrate specificity and activity of yeast Nha antiporters.