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

Abstract

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.It has been shown that the addition of potassium ions to a yeast cell suspension produces an immediate decrease in the levels of ATP and an increase in ADP and P i (20). These changes, which can be also produced by an increase of the medium pH (21), were later postulated to be due to an increased activity of a membrane H ϩ -ATPase which was activated by high pH or also by potassium ions when the medium pH was low. Further studies proved this hypothesis to be true (19) and valid also for many other fungi and other systems (11). Studies of this system allowed for the first time the cloning and sequencing of the gene encoding the plasma membrane H ϩ

Trehalose-Mediated Inhibition of the Plasma Membrane H + -ATPase from Kluyveromyces lactis : Dependence on Viscosity and Temperature

“…The estimated kinetic parameters for the enzyme are listed in Table 1. In the absence of trehalose, these parameters were similar to those reported elsewhere for the H ϩ -ATPases from Neurospora crassa and Saccharomyces cerevisiae (29,33), as expected from the high degree of identity observed in the gene (PMA1) coding for the plasma membrane H ϩ -ATPase in these organisms (27,31). Trehalose inhibited the H ϩ -ATPase, mainly through a decrease in V max (Table 1).…”

Reactive Cysteines of the Yeast Plasma-Membrane H+-ATPase (PMA1)

“…Six of the cysteines found in the S. cerevisiae enzyme (Cys-148 in membrane segment 2; Cys-376, Cys-409, Cys-472, and Cys-532 in the central catalytic region; and Cys- 867 in membrane segment 10) have been completely conserved throughout the fungal PMA enzymes for which sequences are currently available (2,37). The remaining three (Cys-221 in the small hydrophilic loop between segments 2 and 3; Cys-312 in segment 3; and Cys-569 toward the end of the central catalytic region) have varied, and additional Cys residues have appeared at as many as four additional sites (in the ATPase from Histoplasma capsulatum).…”

The KlTrk1 gene encodes a low affinity transporter of the K+ uptake system in the budding yeast Kluyveromyces lactis

“…Escherichia coli strain DHα5 was used to propagate shuttle vectors and recombinant plasmids. Plasmids KEp6 and YEp352 (Miranda et al ., 1995) were used to transfect K. lactis and S. cerevisiae strains, respectively. YIp352 was derived from YEp352 by removing the 2μ replication origin and used as an integrative vector for gene disruption.…”

“…Its life cycle resembles that of the S. cerevisiae and shares high similarities in haploid and diploid vegetative growth and in sexual reproduction. K. lactis mutants, defective in potassium uptake, have been isolated (Brunner et al ., 1982; Miranda et al ., 1995) and one of these led to cloning and characterizing the plasma membrane H + ‐ATPase‐encoding gene. A mutation that caused low H + ‐pumping activity was used to demonstrate the direct functional relationship between the membrane potential generated by the H + ‐ATPase and the K + uptake system in this yeast (Miranda et al ., 1995).…”