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 Pro145 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 Pro145 with glycine resulted in a ZrSod2-22p with extremely low activity only for Na+, and the exchange of a charged residue (Asp, Lys) for Pro145 completely stopped the activity. Mutagenesis of the corresponding proline in the S. cerevisiae Nha1 antiporter (Pro146) 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.
Kinclova-Zimmermannova
Olga
O
Department of Membrane Transport, Institute of Physiology AS CR, Prague 4, 142 20, Czech Republic. kinclova@biomed.cas.cz
Zavrel
Martin
M
Sychrova
Hana
H
D000483:Alleles; D000595:Amino Acid Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics* / Q000502:physiology*; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005656:Fungal Proteins / Q000235:genetics* / Q000502:physiology*; D005998:Glycine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011188:Potassium / Q000737:chemistry / Q000378:metabolism; D011392:Proline / Q000737:chemistry*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012492:Salts / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers; D013045:Species Specificity; D013379:Substrate Specificity; D013997:Time Factors; D014466:Ultraviolet Rays; D020068:Zygosaccharomyces / Q000378:metabolism*
D002352:Carrier Proteins; D002412:Cations; D005656:Fungal Proteins; C434280:SOD22 protein, Zygosaccharomyces; D012492:Salts; D017923:Sodium-Hydrogen Exchangers; D011392:Proline; D011188:Potassium; D005998:Glycine