Molecular cloning of the plasma membrane H(+)-ATPase from Kluyveromyces lactis: a single nucleotide substitution in the gene confers ethidium bromide resistance and deficiency in K+ uptake.
A Kluyveromyces lactis strain resistant to ethidium bromide and deficient in potassium uptake was isolated. Studies on the proton-pumping activity of the mutant strain showed that a decreased H(+)-ATPase specific activity was responsible for the observed phenotypes. The putative K. lactis PMA1 gene encoding the plasma membrane H(+)-ATPase was cloned by its ability to relieve the potassium transport defect of this mutant and by reversing its resistance to ethidium bromide. Its deduced amino acid sequence predicts a protein 899 residues long that is structurally colinear in its full length to H(+)-ATPases cloned from different yeasts, except for the presence of a variable N-terminal domain. By PCR-mediated amplification, we identified a transition from G to A that rendered the substitution of the fully conserved methionine at position 699 by isoleucine. We attribute to this amino acid change the low capacity of the mutant H(+)-ATPase to pump out protons.
Miranda
M
M
Departamento de Microbiología, Universidad Nacional Autónoma de México, D.F., Mexico.
Ramírez
J
J
Peña
A
A
Coria
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial; D004996:Ethidium / Q000494:pharmacology*; D005800:Genes, Fungal; D007716:Kluyveromyces / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011188:Potassium / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid
C434364:PMA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases; D004996:Ethidium; D011188:Potassium