Molecular and biochemical characterization of the Dio‐9‐resistant pma1‐1 mutation of the H⁺‐ATPase from Saccharomyces cerevisiae

Abstract

The plasma-membrane H +-ATPase gene P M A l was sequenced in four Dio-9-resistant strains of Saccharomyces cerevisiae, isolated independently. The same amino acid substitution Ala608 + Thr was found in the four mutated strains. The mutant ATPase activity was decreased while the K,,, value for MgATP was increased. The ATPase efficiency ( V/Km) of the mutant was reduced by a factor of 25 under acid conditions (pH 5.9, and by a factor of 10 at physiological pH (pH 6.6). The mutation also strongly reduces the inhibition by vanadate of ATPase activity, suggesting that the altered amino acid is involved in phosphate binding and/or in the El -E2 transition. Mutations affecting the plasma-membrane H'-ATPase of Saccharomyces cerevisiae were initially obtained by selecting mutants resistant to the Dio-9, an antibiotic of unknown structure [15, 161. Four allelic nuclear mutants defining the P M A l locus were modified in several ATPase catalytic properties including low specific activity, high K, for MgATP and strong vanadate resistance [15]. Additional pmal mutants have been selected in Schizosaccharomyces pombe using , and in S. cerevisiae using the aminoglycoside antibiotic hygromycin B [18]. In each case, the mechanism of resistance appears to be linked to a reduced ATPase activity, probably leading to a decrease in drug uptake [19, 201 Enzyme. ATPase (EC 3.6.1.3).In this paper we present the molecular and biochemical characterization of the four original Dio-9-resistant pmal mutants of S. cerevisiae. In these mutants, the Ala608 + Thr substitution leads to marked kinetic modifications of the plasma-membrane H+-ATPase activity, including decreased sensitivity to vanadate. MATERIALS AND METHODS Yeast strainsThe four pmal-1 mutant strains, MG2129, MG2130, MG2131 and MG2132, are isogenic to the wild-type strain C1278b [15]. Media and growth conditionsYeast strains were grown at 30°C in YD medium containing 2% yeast extract (Difco) and 2% glucose. Escherichia coli strains were grown at 37°C in a conventional LB medium supplemented with 100 pg/ml ampicillin, where appropriate. Solid media were supplemented with 2% Bactor-Agar (Difco). Construction and screening of genomic librariesGenomic DNA was isolated from the wild-type C1278b and thepmal-1 mutant strains. DNA was digested to completion with XhoI and HindIII, then ligated into the Sun-HindIII-cut pTZ19U. Ligation products were amplified in E. coli RRI, and approximatively 15 000 colonies were transferred to nitrocellulose filters. Screening was made using a nicktranslated Hind111 -XbaI fragment, containing the whole P M A l gene from the S. cerevisiae strain IL125-2B [16] as a probe.Filters were washed for 2 h at 42°C (1 M NaCl, 1 mM EDTA, 50 mM Tris/HCl, pH 8.0, 0.1% SDS), then prehybridized for 2 h at 42°C in 50% formamide, 5 x Denhardt's solution, 0.1 O h SDS and 0.75 M NaC1,0.04 M sodium dihydrogen phosphate, 5 mM EDTA, pH 7.4. Hybridization was carried

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

“…The methionine is located contiguous to an also fully conserved arginine which has been pinpointed as an interesting target for site-directed mutagenesis (38). The effect of the substitution of methionine 669 agrees very well with changes in activity found by substitutions of amino acid residues in the large cytoplasmic hydrophilic loop (5,12,27,30,37). However, further biochemical studies of this mutant are needed to determine whether the decrease in activity of the ATPase is due to defective binding of ATP and phosphorylation and/or to what extent, if any, the enzyme conformation is altered.…”

Activation of H+-ATPase by glucose in Saccharomyces cerevisiae involves a membrane serine protease

“…It could be possible that tubulin association to Pma1p is modified with no complete detachment. If one consider this last point, and the fact that the Km ATP of Pma1p changes from 2 mM to 1 mM and V max is increased a 100% when pH varies from 7 to 6 [42], the delay in Pma1p activation observed for lpx1Δ mutant in Fig. 6A could be generating the decrease in cytosolic pH that modifies the kinetic parameters that enhance Pma1p activity, explaining the faster rate observed in lpx1Δ cells at time 300 s in Fig.…”