Contribution of 18O Technology to the Mechanism of the H+-ATPase from Yeast Plasma Membrane

Amory, Antoine; Goffeau, André; McIntosh, David B.; Boyer, Paul D.

doi:10.1016/b978-0-12-152824-9.50047-2

Cited by 10 publications

(12 citation statements)

References 21 publications

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“…On the basis of these results the previous reaction schemes for ATP hydrolysis by the H+-ATPase from yeast (Amory et al, 1982;Nakamoto and Slayman, 1989;Goffeau and Green, 1990) can be simplified as shown in Fig. 8.…”

Section: Discussionmentioning

confidence: 94%

“…One has the ability to bind ATP and is phosphorylated by the y-phosphate of the nucleotide. The other does not bind ATP and is phosphorylated by Pi in the presence of magnesium (Amory et al, 1982). ATP hydrolysis, catalyzed by P-type ATPases, is strongly inhibited by vanadate through a non-competitive mechanism (Dufour et al, 1980;Borst-Pauwels and Peters, 1981).…”

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confidence: 99%

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The plasma membrane H⁺ ‐ATPase from yeast

Wach

Gräber

1991

European Journal of Biochemistry

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The H+-ATPase from the plasma membrane of Saccharnmyces cerevisiae was isolated and purified. The rate of ATP hydrolysis and ATP binding was measured as a function of pH and the effect of the vanadate and erythrosine B inhibitors was investigated.The pH dependence of the rate of ATP hydrolysis forms a bell-shaped curve with a maximum at pH 6 and half-maximal rates at pH 5.0 and 7.4. Only the pH dependence between pH 6 and pH 7.6 is reversible. Above pH 7.6 and below pH 5.5, denaturation of the isolated enzyme is observed. The rate of ATP binding shows the same pH dependency as that of ATP hydrolysis. Both pH dependencies can be described by the dissociation of a monovalent acidic group with a pK of 7.4. It is concluded that the enzyme must be protonated before ATP binding.Vanadate does not inhibit ATP binding, ADP release or Pi release at concentrations where complete inhibition of ATP hydrolysis is observed. It is concluded that vanadate inhibits a step of the reaction cycle which occurs after Pi release. In contrast, erythrosine B inhibits ATP binding and thus affects the first step of the reaction cycle.The plasma membrane Hf-ATPase is an integral membrane protein of yeast cells. The enzyme hydrolyzes ATP and transports protons from the cytosol to the extracellular medium. It was proposed that the H+-ATPase plays an important role in controlling several cellular functions such as nutrient uptake, intracellular pH and cell growth [see reviews by Goffeau and Slayman (1981);Serrano (1988); Goffeau and Green (1990); Serrano (1989)l.The plasma membrane H+-ATPase from yeast belongs to the family of P-type ATPases (Pedersen and Carafoli, 1987). These ATPases are characterized by a covalent phosphorylated intermediate which is formed during the catalytic cycle by transfer of the y-phosphate of ATP to an aspartic acid residue (Amory et al., 1980). The P-type ATPases exist in two major functional states. One has the ability to bind ATP and is phosphorylated by the y-phosphate of the nucleotide. The other does not bind ATP and is phosphorylated by Pi in the presence of magnesium (Amory et al., 1982). ATP hydrolysis, catalyzed by P-type ATPases, is strongly inhibited by vanadate through a non-competitive mechanism (Dufour et al., 1980;Borst-Pauwels and Peters, 1981). It was suggested that vanadate acts as a transition state analogue of phosphate (Cantley et al., 1978; Willsky, 1979). Erythrosine B inhibits ATP hydrolysis catalyzed by the P-type Mg2 + ATPase from radish microsomes (Cocucci and Marre, 1984). Erythrosine B is an eosin derivative and it is assumed to bind at the ATP binding site, i.e. its mechanism is similar to that found for eosin and the Na' /K+-ATPase (Skou and Esmann, 1981).Correspondence to P. Graber, Biologisches Institut, Universitat Stuttgart, Pfaffenwaldring 57, W-7000 Stuttgart 80, Federal Repbulic of GermanyEnzyme. ATPase or adenosine triphosphatase (EC 3.6.1.3).In this work we want to identify parts of the reaction sequence of the catalytic cycle of H+-ATPase, and determine (a) in which step pro...

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Section: Discussionmentioning

confidence: 94%

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confidence: 99%

The plasma membrane H⁺ ‐ATPase from yeast

Wach

Gräber

1991

European Journal of Biochemistry

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“…This is in agreement with the observation in the H+-ATPase of a high K, for MgATP, well above the E . MgATP dissociation constant [35] and the very high K, for the phosphorylation by magnesium phosphate [36]. The fact that the turnover of the H +-ATPase is limited by steps different from those limiting the Ca2+-ATPase could also be reflected by the different inhi- Fig.…”

Section: Discussionmentioning

confidence: 99%

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

Dyck

Petretski

Wolosker

et al. 1990

European Journal of Biochemistry

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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

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“…One ofthese pumps is localized on the tonoplast membrane and is similar to other vacuolar type ATPases being inhibited by NEM and nitrate, but insensitive to vanadate (7,28). The other pump is believed to be localized on the plasma membrane and similar to other E1-E2 type ATPase in forming an aspartyl phosphate intermediate, being sensitive to vanadate and utilizing Mg-ATP as substrate (1,4,6,7,25,28). Transport ATPases of the El -E2 type have been shown to exist in at least two different conformational states depending on the ligands bound to the enzyme (1,25).…”

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confidence: 94%

“…The other pump is believed to be localized on the plasma membrane and similar to other E1-E2 type ATPase in forming an aspartyl phosphate intermediate, being sensitive to vanadate and utilizing Mg-ATP as substrate (1,4,6,7,25,28). Transport ATPases of the El -E2 type have been shown to exist in at least two different conformational states depending on the ligands bound to the enzyme (1,25). These conformational states have been deduced by changes in susceptibility to proteolytic degradation ( 19 and references cited therein) and fluorescence of aromatic amino acids within the protein (14,16) and covalently bound probes (15).…”

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Kinetic Analysis of Proton Transport by the Vanadate-Sensitive ATPase from Maize Root Microsomes

Bräuer¹,

Tu²,

Hsu³

et al. 1989

Plant Physiol.

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Proton transport by the nitrate-insensitive, vanadate-sensitive ATPase in KI-washed microsomes and reconstituted vesicles from maize (Zea mays L.) roots was followed by changes in acridine orange absorbance in the presence of either KNO3 or KCI. Data from such studies obeyed a kinetic model in which net proton transport by the pump is the difference between the rate of proton transport by the action of the ATPase and the leak of protons from the vesicles in the direction opposite from the pump. After establishing the steady state proton gradient, the rate of return of transported protons was found to obey first-order kinetics when the activity of the ATPase was completely and rapidly stopped. The rate of return of these protons varied with the quencher used. When the substrate Mg:ATP was depleted by the addition of either EDTA or hexokinase, the rate at which the proton gradient collapsed was faster than when vanadate was used as the quencher. These trends were independent of the anion accompanying the K and the transport assay used.Membranes from maize roots have been shown to contain at least two proton transporting ATPases (6,28). One ofthese pumps is localized on the tonoplast membrane and is similar to other vacuolar type ATPases being inhibited by NEM and nitrate, but insensitive to vanadate (7,28). The other pump is believed to be localized on the plasma membrane and similar to other E1-E2 type ATPase in forming an aspartyl phosphate intermediate, being sensitive to vanadate and utilizing Mg-ATP as substrate (1,4,6,7,25,28). Transport ATPases of the El -E2 type have been shown to exist in at least two different conformational states depending on the ligands bound to the enzyme (1, 25). These conformational states have been deduced by changes in susceptibility to proteolytic degradation ( 19 and references cited therein) and fluorescence of aromatic amino acids within the protein (14,16) and covalently bound probes (15). It has been postulated that the changes in protein structure are essential for ion movement (25), because the conformation of the E 1-E2 type ATPase is affected by binding of transported cation (14).Characterization of proton transport by the vanadate-sensitive pump from maize roots has been slowed because of difficulties in purifying plasma membranes with competent transport activities. Problems in isolating these membranes arise from the abundance of proteases in membrane fractions (8) and the presence of lipolytic activities which affect membrane integrity (3). Recent advances in purification of membranes from roots have allowed isolation of vesicles with vanadate-sensitive proton transport (6, 7, 10). Additionally, several reconstitution protocols have been developed to insert the vanadate-sensitive ATPase into liposomes (3,26).In a recent article (28), a kinetic model for describing proton transport by the tonoplast ATPase was proposed. This model quantifies the overall process of proton transport by simultaneously considering the pumping and the leakage of protons from membra...

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Contribution of 18O Technology to the Mechanism of the H+-ATPase from Yeast Plasma Membrane

Cited by 10 publications

References 21 publications

The plasma membrane H⁺ ‐ATPase from yeast

The plasma membrane H⁺ ‐ATPase from yeast

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

Kinetic Analysis of Proton Transport by the Vanadate-Sensitive ATPase from Maize Root Microsomes

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Contribution of 18O Technology to the Mechanism of the H+-ATPase from Yeast Plasma Membrane

Cited by 10 publications

References 21 publications

The plasma membrane H+ ‐ATPase from yeast

The plasma membrane H+ ‐ATPase from yeast

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

Kinetic Analysis of Proton Transport by the Vanadate-Sensitive ATPase from Maize Root Microsomes

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The plasma membrane H⁺ ‐ATPase from yeast

The plasma membrane H⁺ ‐ATPase from yeast

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