Identification of Conserved Prolyl Residue Important for Transport Activity and the Substrate Specificity Range of Yeast Plasma Membrane Na+/H+ Antiporters

Abstract

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. 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. Na+/H+ antiporters are ubiquitous membrane proteins found in cytoplasmic and organelle membranes in most organisms, including bacteria, fungi, plants, and animals. They play crucial roles in the homeostasis of alkali metal cations, maintenance of intracellular pH, and cell volume (1Padan E. Venturi M. Gerchman Y. Dover N. Biochim. Biophys. Acta. 2001; 1505: 144-157Crossref PubMed Scopus (285) Google Scholar, 2Orlowski J. Grinstein S. Pflugers. Arch. 2004; 447: 549-565Crossref PubMed Scopus (548) Google Scholar, 3Sychrova H. Physiol. Res. 2004; 53: S91-S98PubMed Google Scholar). The model yeast Saccharomyces cerevisiae possesses three genes encoding monovalent cation/H+ antiporters: NHA1 (4Prior C. Potier S. Souciet J.L. Sychrova H. FEBS Lett. 1996; 387: 89-93Crossref PubMed Scopus (149) Google Scholar), NHX1 (5Nass R. Cunningham K.W. Rao R. J. Biol. Chem. 1997; 272: 26145-26152Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), and KHA1 (6Ramirez J. Ramirez O. Saldana C. Coria R. Pena A. J. Bacteriol. 1998; 180: 5860-5865Crossref PubMed Google Scholar). Whereas the protein products of NHX1 and KHA1 genes are localized in intracellular compartments (7Nass R. Rao R. J. Biol. Chem. 1998; 273: 21054-21060Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 8Maresova L. Sychrova H. Mol. Microbiol. 2005; 55: 588-600Crossref PubMed Scopus (77) Google Scholar), the Nha1p belongs to the unique clade of fungi-specific plasma membrane Na+/H+ antiporters (9Kinclova O. Ramos J. Potier S. Sychrova H. Mol. Microbiol. 2001; 40: 656-668Crossref PubMed Scopus (98) Google Scholar, 10Brett C.L. Donowitz M. Rao R. Am. J. Physiol. Cell Physiol. 2005; 288: C223-C239Crossref PubMed Scopus (433) Google Scholar). Nha1p uses a protonmotive force generated by the plasma membrane H+-ATPase to mediate the efflux of Na+, Li+, K+, and Rb+ from cells (9Kinclova O. Ramos J. Potier S. Sychrova H. Mol. 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Yeast plasma membrane Na+/H+ antiporters including a amino a transmembrane with transmembrane and a acid and the of the transmembrane and are highly conserved the antiporter are their and amino acid are O. Potier S. Sychrova H. Microbiology. 2002; PubMed Scopus Google Scholar). the transport specificity and transport of yeast Na+/H+ antiporters their in an S. cerevisiae strain antiporters divided in two distinct antiporters with substrate specificity only for Na+ and and in and a of antiporters the transport of at alkali metal cations, the of cations, play a in cell of intracellular pH, and cell O. Potier S. Sychrova H. Microbiology. 2002; PubMed Scopus Google Scholar). ScNha1p and found to in a efflux a of cell to the of pH, that both antiporters play a in the of intracellular O. Potier S. Sychrova H. FEBS Lett. 2001; 504: 11-15Crossref PubMed Scopus (24) Google Scholar). of intracellular proved that the of in the of H. J. Pena A. Microbiol. Lett. PubMed Scopus Google Scholar, C.L. S. Rao R. Mol. Biol. 2005; PubMed Scopus Google Scholar). the antiporter is in the of the cell E. J. Calero F. Ramos J. J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), and in the cell to (9Kinclova O. Ramos J. Potier S. Sychrova H. Mol. Microbiol. 2001; 40: 656-668Crossref PubMed Scopus (98) Google Scholar, M. K. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). of from sorbithophila (16Banuelos M.A. Ramos J. Calero F. Braun V. Potier S. Yeast. 2002; 19: 1365-1372Crossref PubMed Scopus (23) Google and C. (19Kamauchi S. Mitsui K. Ujike S. Haga M. Nakamura N. Inoue H. Sakajo S. Ueda M. Tanaka A. H. J. 2002; PubMed Scopus Google the of antiporters with a substrate of antiporters the two their of protein S. cerevisiae Nha1p the with the rouxii The of and transport of an alkali metal by the yeast antiporter is The substrate specificity of antiporters to only by the of their transmembrane and the of the ScNha1p to and transport (9Kinclova O. Ramos J. Potier S. Sychrova H. Mol. Microbiol. 2001; 40: 656-668Crossref PubMed Scopus (98) Google Scholar). of the antiporter amino acid residues important for L. FEBS Lett. 1998; PubMed Scopus Google Scholar, C. Young P.G. L. Mol. Cell PubMed Scopus Google Scholar, L. Mol. Cell 2005; Google Scholar). residues are conserved in the antiporters of and of an low in their L. FEBS Lett. 1998; PubMed Scopus Google Scholar). The of residues in the to for the of the rouxii antiporter Y. Y. Tsuji H. Tamai Y. Microbiol. Lett. 2002; PubMed Scopus Google Scholar), and of conserved residues and to the transport activity in C. albicans and S. cerevisiae Nha1p (17Soong T.W. Yong T.F. Ramanan N. Wang Y. Microbiology. 2000; 146: 1035-1044Crossref PubMed Scopus (35) Google Scholar, E. J. Calero F. Ramos J. J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). in the of the efflux activity was and was residue mutation E. J. Calero F. Ramos J. J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). this to identify the amino acid residues that are responsible for the determination of the Na+/H+ antiporter substrate specificity to and a collection of mutants of the rouxii Sod2-22 antiporter K+) that improved the S. cerevisiae tolerance to KCl was isolated. Several independent ZrSod2-22 mutants contained the replacement of the residue 145 with an amino acid containing a hydroxyl group (Ser, Thr). The Pro145 the fifth transmembrane domain is highly conserved of the two subfamilies. Site-directed mutagenesis of Pro145 proved that the of an amino acid residue containing a hydroxyl group at this position is enough to enable ZrSod2-22p to transport K+, the of residues the antiporter from of a non-polar amino acid residue important for the activity and substrate specificity of yeast Nha antiporters an important the transport of yeast plasma membrane Na+/H+ antiporters. and S. cerevisiae strain to O. Ramos J. Potier S. Sychrova H. Mol. Microbiol. 2001; 40: 656-668Crossref PubMed Scopus (98) Google and 11Banuelos M.A. Sychrova H. Bleykasten-Grosshans C. Souciet J.L. Potier S. Microbiology. 1998; 144: 2749-2758Crossref PubMed Scopus (200) Google was in this for Yeast cells at with and by in Microbiology. Scholar). with the S. cerevisiae NHA1 and the a from 15Kinclova O. Potier S. Sychrova H. J. Biotechnol. 2001; 88: 151-158Crossref PubMed Scopus (42) Google was cloned of the R. M. 1995; PubMed Scopus Google to the for and the corresponding of C. J. J. PubMed Scopus Google Scholar). was a for in the mutagenesis of the of from mutated and cloned the A. Yeast. PubMed Scopus Google Scholar). the mutagenesis of the S. cerevisiae the with was from the (9Kinclova O. Ramos J. Potier S. Sychrova H. Mol. Microbiol. 2001; 40: 656-668Crossref PubMed Scopus (98) Google and cloned the corresponding of in the was cloned the the The was by the from the corresponding of the and the of J. T. Scholar). of cells containing mutagenized by in independent of the a yeast was of to and cells The was from with for to cell The that of at The cell of mutagenesis was with at for to to a of mutagenesis of the by was the Mutagenesis The was from in the with and of in the was by the independent with a of and The was with at to the and to the the of products with the of to the cells a in Microbiology. Scholar). containing with to and in from corresponding to with at for to to a of Site-directed and the Site-directed Mutagenesis mutation two containing the corresponding The of mutation was confirmed by of by yeast cells by of of with an of KCl with and (9Kinclova O. Ramos J. Potier S. Sychrova H. Mol. Microbiol. 2001; 40: 656-668Crossref PubMed Scopus (98) Google Scholar). was for in to an and To the efflux of Na+, cells for in at with (9Kinclova O. Ramos J. Potier S. Sychrova H. Mol. Microbiol. 2001; 40: 656-668Crossref PubMed Scopus (98) Google Scholar). efflux from cells was in of was to with and was to the to and with KCl in the of cells at and the intracellular of was by (9Kinclova O. Ramos J. Potier S. Sychrova H. Mol. Microbiol. 2001; 40: 656-668Crossref PubMed Scopus (98) Google Scholar, R. A. FEBS Lett. PubMed Scopus Google Scholar). was three and are To identify amino acid residues in the of for Sod2-22 mutants of To the for the ZrSod2-22p was in the S. cerevisiae strain is to of the of the and and Nha1 the of the rouxii antiporter the tolerance of cells to and The tolerance the of antiporter from a from a The to of cells ZrSod2-22p was low in cells with antiporter the the of an antiporter efflux resulted in a in tolerance This to the and for mutants with substrate specificity to transport of and by S. cerevisiae cells rouxii S. cerevisiae NHA1 in a the mutagenesis by The is for the mutagenesis of genes genes in cells from low cells with the containing the cells containing This was mutants at KCl in KCl from independent and of proved a tolerance to in three three with a of KCl to cells with the ZrSod2-22p only that three contained the with a of proline 145 for 145 is to the fifth transmembrane domain and is conserved in of yeast plasma membrane Na+/H+ antiporters to that residues important for the of a substrate in the protein of ZrSod2-22p only and ScNha1p Na+ and K+) the substrate specificity of ZrSod2-22p of the of a highly conserved residue was of ZrSod2-22 mutants by and acid of the in a Mutagenesis by only of ZrSod2-22p been by the mutagenesis of the by was to the of ZrSod2-22p mutants independent with corresponding to a mutation of to the mutated alleles a collection of was with of a for their tolerance to and KCl and divided the of the of The corresponding from the group and cells to the containing mutated for the their in cells the tolerance to that independent ZrSod2-22 mutants with of proline 145 for an amino acid residue with a hydroxyl group by this of residue for Pro145 for and The of amino acid residues mutated in and are in the model of Sod2-22 protein The contained three in a amino acid of Pro145 for and two in for and the the protein was to the by The of improved the tolerance of cells to KCl and to The mutation in two amino acid Pro145 was with and with cells a of KCl cells with in KCl tolerance was in three by a in tolerance from the to and to a of the transport of the three mutated alleles cloned the and to the substrate specificity of mutated of the of was in containing of and with the a cells the ZrSod2-22p and The of ScNha1p cells with an tolerance to cations, cells containing the only with of and with in cells containing mutated of ZrSod2-22p of and KCl cells with the The of mutated ZrSod2-22 proteins from the of antiporters the of and cells the from a low containing to and KCl to from a the and KCl Whereas the of KCl was the a of cells mutated ZrSod2-22 in with ScNha1p was the first of cells with the ZrSod2-22p with and the to of the three mutants improved the of strain with This that the of the to transport was to a the of the to transport is a in the of J. A. J. PubMed Scopus Google Scholar). mutants improved the tolerance of to cells to with of with mutated ZrSod2-22p to the of cells with the ZrSod2-22p cells containing the in that three ZrSod2-22p mutants with of Pro145 and and transport only and and The transport activity of Na+/H+ antiporters is by the of the plasma the A. Biochim. Biophys. Acta. PubMed Scopus (45) Google Scholar, Physiol. 1995; PubMed Scopus Google Scholar). have that the and tolerance of S. cerevisiae cells the ZrSod2-22p decreased with O. Potier S. Sychrova H. Microbiology. 2002; PubMed Scopus Google Scholar). to and and with that the and tolerance of cells containing mutated ZrSod2-22p was the pH, the at and at This that the of the transport activity of ZrSod2-22p was by the by that mutated ZrSod2-22 proteins are to their the efflux from cells the three mutated in to the of with to and the was for the a efflux of of in and cells with the the ZrSod2-22p the the intracellular of decreased of both in cells antiporter and in cells containing the ZrSod2-22p the are in the strain that the in the activity of was in a efflux of was from cells containing the mutated and of and The efflux by the mutated was for the the that the by mutated ZrSod2-22 low to enable the of cells in the of of KCl efflux confirmed that both of ZrSod2-22 mutants with a mutation of Pro145 to in to the transport of K+. was from and efflux that the the protein with the Pro145 is the highly conserved of the fifth transmembrane domain of yeast plasma membrane Na+/H+ antiporters an at the of Pro145 in the determination of the Na+/H+ antiporter substrate specificity and the of for an amino acid residue with a hydroxyl Site-directed Mutagenesis of Pro145 in that the of ZrSod2-22p to transport is to the of an amino acid residue containing the hydroxyl group of the residue at position mutants of the in the for Pro145 was by the encoding amino with a hydroxyl group (Ser, a amino acid and charged amino (Asp, alleles cloned a in and to mutants from mutagenesis ZrSod2-22 antiporters of Pro145 to charged amino acid residues improved the tolerance of cells to to This the of activity of the The of the in cells the tolerance of cells to to KCl to low transport activity of the mutated cells ZrSod2-22p with a of Pro145 for with KCl The and to the tolerance of cells to to a the ZrSod2-22 antiporter was for the mutants and the ZrSod2-22p with of Pro145 for cells with tolerance the in confirmed by of and with was found in cells the ZrSod2-22 mutants Pro145 was mutated to ZrSod2-22 antiporters Pro145 was for the efflux of from cells with to for mutants and and the intracellular of Na+ in S. cerevisiae cells is at in cells with the of Na+ of for the efflux cells in a of containing KCl to Na+ The of from cells was for the strain the intracellular of Na+ was the the by ZrSod2-22 antiporters. efflux of Na+ was from cells ZrSod2-22p with of Pro145 to to the of cells to the that the was to the tolerance of cells of Na+ by this protein was the cells with a tolerance to the ZrSod2-22p the of Na+ efflux the an efflux of from cells with a of transport for the ZrSod2-22 antiporter The efflux from cells the was from cells with the ZrSod2-22p the to a tolerance of cells to Site-directed mutagenesis of Pro145 in the ZrSod2-22 antiporter proved that the of an amino acid residue containing a hydroxyl group at this position is enough to enable ZrSod2-22p to transport and that the is the residue the hydroxyl a group in that the mutation in the a in substrate specificity to and transport Mutagenesis of in S. cerevisiae the residue in the fifth transmembrane domain is conserved in the of yeast plasma membrane Na+/H+ antiporters The that ZrSod2-22p the to transport by mutation of the conserved Pro145 to the of this mutation the substrate specificity and transport activity of the S. cerevisiae Nha1 this the mutation in ScNha1p to Pro145 in ZrSod2-22p was the The antiporter was in cells from a the of the NHA1 and transport activity was with the corresponding ScNha1p the the substrate specificity of antiporter was in containing of Na+, Li+, K+, and Rb+ antiporter a The mutation of to in ScNha1p the cell tolerance to containing the antiporter with KCl cells the ScNha1p the the cells with tolerance the the of cells with the cells the ScNha1p with the with cells containing the cells with the ScNha1p with that the mutation of to the and transport of three of the ScNha1p Na+, Li+, and and efflux activity by the antiporter was the for the of efflux by ZrSod2-22 that the of efflux from cells the was from cells with the ScNha1p of in the that the antiporter with mutation cells with the of KCl tolerance the ScNha1p This that to the of Sod2-22 the efflux activity was to a cell tolerance of in of Na+ from cells with was with cells with the This to the tolerance of cells with and is that the intracellular of Na+ at the of the a of cells in with cells with contained a of Na+ cells antiporter cells with the ScNha1p of the Na+ efflux activity of was low the substrate was in cells with the decreased of Na+ and in cells with from of to the plasma To this the was at with the protein and in The the and efflux activity to the cells by the mutated of the corresponding to the plasma membrane for the antiporter was for the ScNha1p (9Kinclova O. 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