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J. Bacteriol., May 1995, 2360-2367, Vol 177, No. 9
Copyright © 1995, American Society for Microbiology

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

M Miranda, J Ramirez, A Pena and R Coria
Departamento de Microbiologia, Universidad Nacional Autonoma de Mexico, D.F., Mexico.

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.

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