Publications

• The complete inventory of the yeast Saccharomyces cerevisiae P-type transport ATPases.

  Catty P., de Kerchove d'Exaerde A., Goffeau A.

  A total of sixteen open reading frames encoding for P-type ATPases have been identified in the complete genome sequence of Saccharomyces cerevisiae. Phylogenetic analysis distinguishes 6 distinct families. Topology predictions, identification of aminoacid sequence motifs and phenotype analysis of ... >> More

  A total of sixteen open reading frames encoding for P-type ATPases have been identified in the complete genome sequence of Saccharomyces cerevisiae. Phylogenetic analysis distinguishes 6 distinct families. Topology predictions, identification of aminoacid sequence motifs and phenotype analysis of the available mutants suggest that these families correspond to ATPases transporting either H+ (2 members), Ca2+ (2 members), Na+ (3 members), heavy metals (2 members), possibly aminophospholipids (5 members including 4 new ones) or unknown substrates (2 new members). It is proposed that the latter family which has homologs in Tetrahymena thermophila, Plasmodium falciparum and Caenorhabditis elegans constitutes a new group called P4-ATPases with characteristic topology and aminoacid signatures. << Less

  FEBS Lett 409:325-332(1997) [PubMed] [EuropePMC]

• A two-gene ABC-type transport system that extrudes Na+ in Bacillus subtilis is induced by ethanol or protonophore.

  Cheng J., Guffanti A.A., Krulwich T.A.

  A transposition mutant of Bacillus subtilis (designated JC901) that was isolated on the basis of growth inhibition by Na+ at elevated pH, was deficient in energy-dependent Na+ extrusion. The capacity of the mutant JC901 for Na(+)-dependent pH homeostasis was unaffected relative to the wild-type st ... >> More

  A transposition mutant of Bacillus subtilis (designated JC901) that was isolated on the basis of growth inhibition by Na+ at elevated pH, was deficient in energy-dependent Na+ extrusion. The capacity of the mutant JC901 for Na(+)-dependent pH homeostasis was unaffected relative to the wild-type strain, as assessed by regulation of cytoplasmic pH after an alkaline shift. The site of transposition was near the 3'-terminal end of a gene, natB, predicted to encode a membrane protein, NatB. NatB possesses six putative membrane-spanning regions at its C-terminus, and exhibits modest sequence similarity to regions of eukaryotic Na+/H+ exchangers. Sequence and Northern blot analyses suggested that natB forms an operon with an upstream gene, natA. The predicted product of natA is a member of the family of ATP-binding proteins that are components of transport systems of the ATP-binding cassette (ABC) or traffic ATPase type. Expression of the lacZ gene that was under control of the promoter for natB indicated that expression of the operon was induced by ethanol and the protonophore carbonylcyanide p-chlorophenylhydrazone (CCCP), and more modestly, by Na+, and K+, but not by choline or a high concentration of sucrose. Restoration of the natAB genes, cloned in a recombinant plasmid (pJY1), complemented the Na(+)-sensitive phenotype of the mutant JC901 at elevated pH and significantly increased the resistance of the mutant to growth inhibition by ethanol and CCCP at pH 7; ethanol was not excluded, however, from the cells expressing natAB, so ethanol-resistance does not result from NatAB-dependent ethanol efflux. Transformation of the mutant with pJY1 did markedly enhance the capacity for Na+ efflux, which was further stimulated by CCCP. In the absence of CCCP, NatAB-mediated Na+ efflux was stimulated by K+. Concomitant NatAB-dependent K+ uptake occurred, as monitored by 86Rb+ uptake; this uptake was inhibited by CCCP and is thus secondary to the primary, electrogenic Na+ efflux. A B. subtilis mutant strain (BsAJ96) in which most of natA and all of natB was replaced by a spectinomycin-resistance-gene cassette exhibited phenotypic properties identical to JC901 Under anaerobic conditions, using a strain of B. subtilis deleted in atp genes encoding the F1F0-ATPase (BD99-A), glucose energized Na+ exclusion in an arsenate-sensitive manner; this exclusion capacity was absent in a strain deleted both in atp and natAB genes (BsAJ96-A). We conclude that NatAB is an inducible, ABC transport system that catalyses ATP-dependent electrogenic Na+ extrusion without mechanistically coupled proton or K+ uptake. This is a novel mode of Na+ extrusion that is hypothesized to play an inducible role in exclusion of cytotoxic Na+ and in the secondary stimulation of K+ uptake, especially when the function of the membrane as an ion-permeability barrier is compromised by agents such as alcohols or uncouplers. << Less

  Mol. Microbiol. 23:1107-1120(1997) [PubMed] [EuropePMC]

• Overexpression of the sodium ATPase of Saccharomyces cerevisiae: conditions for phosphorylation from ATP and Pi.

  Benito B., Quintero F.J., Rodriguez-Navarro A.

  The ENA1 gene of Saccharomyces cerevisiae encodes a putative ATPase necessary for Na+ efflux. Plasma membranes and intracellular membranes of a yeast strain overexpressing the ENA1 gene contain significant amounts of ENA1 protein. Consequences of the overexpression with reference to the wild-type ... >> More

  The ENA1 gene of Saccharomyces cerevisiae encodes a putative ATPase necessary for Na+ efflux. Plasma membranes and intracellular membranes of a yeast strain overexpressing the ENA1 gene contain significant amounts of ENA1 protein. Consequences of the overexpression with reference to the wild-type strain are: (1) a 5-fold higher content of the ENA1-protein in plasma membranes; (2) lower Na+ and Li+ effluxes; (3) slightly higher Na+ tolerance; and (4) much higher Li+ tolerance. The ENA1-specific ATPase activity in plasma membrane preparations of the overexpressing strain was low, but an ENA1 phosphoprotein was clearly detected when the plasma membranes were exposed to ATP in the presence of Na+ or to Pi in the absence of Na+. The characteristics of this phosphoprotein, which correspond to the acyl phosphate intermediaries of P-type ATPases, the absolute requirement of Na+ or other alkali cations for phosphorylation, and the Na+ and pH dependence of phosphorylation from ATP and Pi suggest that the product of the ENA1 gene may be a Na,H-ATPase, which can also pump other alkali cations. The role of the intracellular membranes structures produced with the overexpression of ENA1 in Na+ and Li+ tolerances and the existence of a beta-subunit of the ENA1 ATPase are discussed. << Less

  Biochim. Biophys. Acta 1328:214-226(1997) [PubMed] [EuropePMC]

• The Bacillus subtilis NatK-NatR two-component system regulates expression of the natAB operon encoding an ABC transporter for sodium ion extrusion.

  Ogura M., Tsukahara K., Hayashi K., Tanaka T.

  A previous microarray analysis suggested that multicopy yccH, encoding a function-unknown response regulator, enhances expression of natAB, which encodes a two-gene ATP-binding cassette transporter involved in the extrusion of sodium ions. The two-component regulatory system YccG-YccH was therefor ... >> More

  A previous microarray analysis suggested that multicopy yccH, encoding a function-unknown response regulator, enhances expression of natAB, which encodes a two-gene ATP-binding cassette transporter involved in the extrusion of sodium ions. The two-component regulatory system YccG-YccH was therefore renamed NatK-NatR. Here, this observation was confirmed by a lacZ fusion analysis using a strain carrying natA-lacZ. Further, in both natK and natR mutants, natA-lacZ expression was completely abolished, indicating that the NatK-NatR system positively regulates the expression of natAB. In a gel retardation analysis, NatR bound to the natA promoter region. Using purified His-tagged NatR, DNase I footprinting analysis of the natA promoter region suggested that a direct repeat of [TTCA(G)CGACA], separated by a 12 bp space, would be recognized by NatR. Deleted and mutagenized promoter regions of natA were analysed using a lacZ fusion, and it was confirmed that the direct repeat is critical for natA activation by NatR. << Less

  Microbiology 153:667-675(2007) [PubMed] [EuropePMC]

• The PMR2 gene cluster encodes functionally distinct isoforms of a putative Na+ pump in the yeast plasma membrane.

  Wieland J., Nitsche A.M., Strayle J., Steiner H., Rudolph H.K.

  We report a structural and functional analysis of the PMR2 gene cluster in yeast. We found that several strains of Saccharomyces cerevisiae contain multiple PMR2 genes repeated in tandem, whereas most phylogenetically related yeasts appear to possess only a single PMR2 gene. This unusual tandem ar ... >> More

  We report a structural and functional analysis of the PMR2 gene cluster in yeast. We found that several strains of Saccharomyces cerevisiae contain multiple PMR2 genes repeated in tandem, whereas most phylogenetically related yeasts appear to possess only a single PMR2 gene. This unusual tandem array of nearly identical genes encodes putative ion pumps involved in Na+ tolerance. Pmr2a and Pmr2b, the proteins encoded by the first two repeats, differ by only 13 amino acid exchanges. Both proteins share localization to the plasma membrane, but represent distinct isoforms of a putative Na+ pump. When expressed under identical conditions in vivo, Pmr2a and Pmr2b cause different tolerances to Na+ and Li+. Finally, we show that the Na+ tolerance mediated through these pumps is regulated by calmodulin via a calcineurin-independent mechanism which activates the Pmr2 ion pumps post-transcriptionally. << Less

  EMBO J. 14:3870-3882(1995) [PubMed] [EuropePMC]