Mercaptoethanol and dithiothreitol decrease the difference of electrochemical proton potentials across the yeast plasma and vacuoar membranes and activate their H<sup>+</sup>‐ATPases

Петров, В. А.; Смирнова, В. В.; Okorokov, Lev A.

doi:10.1002/yea.320080803

Cited by 16 publications

(8 citation statements)

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“…Therefore, the decrease in ΔpH and relative stability of ΔΨ support the assumption of proton membrane permeabilization, linked to reducing ORP environment. Similar observations and hypotheses had previously been proposed for Saccharomyces carlsbergensis [29].…”

Section: Discussionsupporting

confidence: 89%

“…The ATPase activity in relation to ORP is constant for all pH values tested. The slight activation observed at a dithiothreitol concentration of 0.5% w/v is due to the chemical nature and the concentration of the compound [29]. Thus, a rising dithiothreitol concentration leads to an increase in ATPase activity, but sodium borohydride utilization does not modify the activity, although it generates a lower ORP (Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Changes in the proton‐motive force in Escherichia coli in response to external oxidoreduction potential

Riondet

Cachon

Waché

et al. 1999

European Journal of Biochemistry

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The pH homeostasis and proton-motive force (Dp) of Escherichia coli are dependent on the surrounding oxidoreduction potential (ORP). Only the internal pH value and, thus, the membrane pH gradient (DpH) component of the Dp is modified, while the membrane potential (DC) does not change in a significant way. Under reducing conditions (E h , 50 mV at pH 7.0), E. coli decreases its Dp especially in acidic media (21% decrease at pH 7.0 and 48% at pH 5.0 for a 850-mV ORP decrease). Measurements of ATPase activity and membrane proton conductance (C H+ m ) depending on ORP and pH have shown that the internal pH decrease is due to an increase in membrane proton permeability without any modification of ATPase activity. We propose that low ORP values de-energize E. coli by modifying the thiol : disulfide balance of proteins, which leads to an increase in the membrane permeability to protons.Keywords: oxidoreduction potential; Escherichia coli; protomotive force; membrane potential; proton membrane conductance.Considerable efforts have been made to understand how cellular metabolism reacts to variations in pH, temperature, pressure and osmotic pressure (reviewed in [1]). However, little information is available on the response of microorganisms to changes in external oxidoreduction potential (ORP), although it is a pH interdependent parameter. Early studies suggested that each species or strain has a preferred redox potential range, within which growth is possible [2±4]. This physicochemical parameter also affects the heat resistance as demonstrated for various microorganisms [5±7]. Certain enzyme activities are controlled by ORP, and modification of this parameter can alter metabolic balances during anaerobic growth in Escherichia coli [8]. This is also true for the synthesis of coproporphyrine by E. coli [9], and the proportion of amino acid synthesized by Clostridium glutamicum [10]. In addition to the regulation of some metabolic pathways, ORP can regulate the expression of genes as shown for the FNR system of E. coli [11]. Recently, Bespalov et al.[12] have shown that E. coli can sense the medium's ORP and swim to a preferred ORP niche by means of redox taxis. This mechanism is consistent with a model in which a change in the proton-motive force (Dp) is the signal that triggers the behavioural response.In this paper, we report that medium's ORP modifies the generation of Dp in E. coli. Of the two parameters that compose Dp (membrane potential, DC, and difference in pH across the membrane, DpH), only DpH decreased when the ORP became more reducing, especially at acidic pH values. The transmembrane electrical potential remained constant. The ATPase activity was not ORP dependent, but that membrane proton conductance (C H+ m ) varied with ORP fluctuations. This deenergization of E. coli was essentially due to an increased membrane permeability to protons. This could be caused by a modification of the thiol : disulfide balance in membrane proteins linked to the medium's ORP. MATERIALS AND METHODSBacterial strains and cultur...

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Section: Discussionsupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Changes in the proton‐motive force in Escherichia coli in response to external oxidoreduction potential

Riondet

Cachon

Waché

et al. 1999

European Journal of Biochemistry

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“…Consistent with this idea, ATPase activity in secretory vesicles is not inhibited by the presence of 10 mM dithiothreitol or 28 mM 2-mercaptoethanol during spheroplast formation. 2 Similarly, Petrov et al (1992) found that 2-mercaptoethanol failed to inhibit the yeast ATPase in isolated plasma membranes. By contrast, the sarcoplasmic reticulum Ca 2ϩ -ATPase-which is known to contain disulfide bonds (see above)-is inactivated by 2-mercaptoethanol and dithiothreitol (Georgoussi and Sotiroudis, 1985;Mutoh et al, 1992;Daiho and Kanazawa, 1994).…”

Section: Fig 3 Hmentioning

confidence: 96%

Site-directed Mutagenesis of the Yeast PMA1 H+-ATPase STRUCTURAL AND FUNCTIONAL ROLE OF CYSTEINE RESIDUES

Петров

Slayman

1995

Journal of Biological Chemistry

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The yeast plasma-membrane H؉ -ATPase contains nine cysteines, three in presumed transmembrane segments (Cys-148, Cys-312, and Cys-867) and the rest in hydrophilic regions thought to be exposed at the cytoplasmic surface (Cys-221, Cys-376, Cys-409, Cys-472, Cys-532, and Cys-569). To gather new functional and structural information, we have studied the yeast ATPase by cysteine mutagenesis. It proved possible to replace seven of the nine cysteines by alanine, one at a time, without any significant decrease in ATP hydrolysis or ATP-dependent proton pumping. In the remaining two cases (Cys-409 and Cys-472), there were small but reproducible effects; the results clearly indicated, however, that no single Cys is required for activity and that, if a disulfide bridge is formed in the yeast ATPase, it does not play an obligatory structural or functional role.Next, multiple mutants were constructed to ask how many Cys residues could be replaced simultaneously while leaving a fully functional enzyme. After substitution of all "membrane" Cys (Cys-148, Cys-312, and Cys-867) together with two non-conserved Cys located in hydrophilic regions (Cys-221 and Cys-569), there were no significant abnormalities in expression (87%) or activity (89% ATP hydrolysis/93% H ؉ pumping) of the mutant protein. Replacement of two additional cysteines (Cys-376 near the phosphorylation site and Cys-532, in or near the ATP-binding site) caused a drop in expression (to 54%), although the corrected hydrolytic and H ؉ pumping activities were still normal. When Cys-472 was also mutated, the corrected activity fell to 44% hydrolysis/47% pumping; finally, substitution of Cys-409 to give a "cysteine-free" ATPase led to a very poorly expressed and poorly active enzyme. Brief exposure of the "onecysteine" and "two-cysteine" ATPases to trypsin revealed a normal pattern of degradation, but there was a slight impairment in the ability of vanadate to protect against proteolysis. Thus, although single Cys replacements are tolerated well by the yeast ATPase, multiple replacements are progressively more harmful, suggesting that they cause small but additive perturbations of protein folding.

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“…cerevisiae proved insensitive to P. fusiformata glycolipids, when the culture-to-culture method was used [6], but was sensitive to a high concentration of puri¢ed glycolipids. The S. cerevisiae cells with a damaged cytoplasmic membrane were shown incapable of acidi¢cation of the external medium in the presence of glucose [10]. 3b).…”

Section: Resultsmentioning

confidence: 99%

“…Acid-i¢cation of the external medium was estimated by a pHmeter in 2 ml of water for 5 min at 30 ‡C after adding 0.1 M glucose [10]. Acid-i¢cation of the external medium was estimated by a pHmeter in 2 ml of water for 5 min at 30 ‡C after adding 0.1 M glucose [10].…”

Section: Acidi¢cation Measurementmentioning

confidence: 99%

ATP leakage from yeast cells treated by extracellular glycolipids of

Kulakovskaya

Golubev

2003

FEMS Yeast Research

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The ustilaginaceous yeast Pseudozyma fusiformata secreted glycolipids which were lethal to many yeasts and fungi more active at pH of about 4.0, and in the temperature range of 20-30 degrees C. Purified glycolipids enhanced non-specific permeability of the cytoplasmic membrane in sensitive cells, which resulted in ATP leakage and susceptibility of the cells to staining with bromocresol purple. Cells of Saccharomyces cerevisiae lost the ability to acidify the medium. Basidiomycetous yeasts were more sensitive to the glycolipids than ascomycetous ones. The minimal effective glycolipid concentration was 0.13 and 0.26 mg ml(-1) for Cryptococcus terreus and Filobasidiella neoformans, while for Candida albicans and Saccharomyces cerevisiae it was 1.0 and 1.6 mg ml(-1).

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Mercaptoethanol and dithiothreitol decrease the difference of electrochemical proton potentials across the yeast plasma and vacuoar membranes and activate their H⁺‐ATPases

Cited by 16 publications

References 72 publications

Changes in the proton‐motive force in Escherichia coli in response to external oxidoreduction potential

Changes in the proton‐motive force in Escherichia coli in response to external oxidoreduction potential

Site-directed Mutagenesis of the Yeast PMA1 H+-ATPase STRUCTURAL AND FUNCTIONAL ROLE OF CYSTEINE RESIDUES

ATP leakage from yeast cells treated by extracellular glycolipids of

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Mercaptoethanol and dithiothreitol decrease the difference of electrochemical proton potentials across the yeast plasma and vacuoar membranes and activate their H+‐ATPases

Cited by 16 publications

References 72 publications

Changes in the proton‐motive force in Escherichia coli in response to external oxidoreduction potential

Changes in the proton‐motive force in Escherichia coli in response to external oxidoreduction potential

Site-directed Mutagenesis of the Yeast PMA1 H+-ATPase STRUCTURAL AND FUNCTIONAL ROLE OF CYSTEINE RESIDUES

ATP leakage from yeast cells treated by extracellular glycolipids of

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Mercaptoethanol and dithiothreitol decrease the difference of electrochemical proton potentials across the yeast plasma and vacuoar membranes and activate their H⁺‐ATPases