Reconstitution of F<sub>1</sub>‐ATPase activity from <i>Escherichia coli</i> subunits α, β and subunit γ tagged with six histidine residues at the C‐terminus

Ekuni, Atsuko; Watanabe, Hikaru; Kuroda, Nozomi; Sawada, Ken; Murakami, Hiroshi; Kanazawa, Hiroshi

doi:10.1016/s0014-5793(98)00395-0

Cited by 6 publications

(4 citation statements)

References 29 publications

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“…The fact that the biomass yield decreased in combination with the reduction in the intracellular energy level is therefore a strong indication that an uncoupled ATPase is active in vivo, as recently found for E. coli (26). Our results are also in agreement with early reconstitution experiments, in which complexes of ␣, ␥, and ␤ (9,10,11), Bacillus strain PS3 (46), and Salmonella enterica serovar Typhimurium (19) were found to have high ATPase activities. The reduction in the intracellular energy level with an increased level of uncoupled F 1 -ATPase did not result in a shift of metabolism from homolactic fermentation to a mixed-acid pattern that would otherwise have provided the cell with extra ATP (14,43).…”

Section: Discussionsupporting

confidence: 92%

Expression of Genes Encoding F ₁ -ATPase Results in Uncoupling of Glycolysis from Biomass Production in Lactococcus lactis

Koebmann

Solem

Pedersen³

et al. 2002

Appl Environ Microbiol

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We studied how the introduction of an additional ATP-consuming reaction affects the metabolic fluxes in Lactococcus lactis. Genes encoding the hydrolytic part of the F 1 domain of the membrane-bound (F 1 F 0 ) H ؉ -ATPase were expressed from a range of synthetic constitutive promoters. Expression of the genes encoding F 1 -ATPase was found to decrease the intracellular energy level and resulted in a decrease in the growth rate. The yield of biomass also decreased, which showed that the incorporated F 1 -ATPase activity caused glycolysis to be uncoupled from biomass production. The increase in ATPase activity did not shift metabolism from homolactic to mixed-acid fermentation, which indicated that a low energy state is not the signal for such a change. The effect of uncoupled ATPase activity on the glycolytic flux depended on the growth conditions. The uncoupling stimulated the glycolytic flux threefold in nongrowing cells resuspended in buffer, but in steadily growing cells no increase in flux was observed. The latter result shows that glycolysis occurs close to its maximal capacity and indicates that control of the glycolytic flux under these conditions resides in the glycolytic reactions or in sugar transport.Lactic acid bacteria are used extensively in the dairy industry, where the production of lactic acid is important for texture, flavor, and preservation purposes. In addition, lactic acid bacteria are also used for industrial lactate production, which has numerous applications, such as cosmetics, cleaning agents, and biodegradable polylactic acid polymers. From an industrial point of view there is great interest in improving the performance of these organisms with respect to both the rate and the yield of lactate production.In spite of the importance of glycolysis for fermentation purposes, it is still not known what controls the glycolytic flux in microbial bioreactors. It has been suggested that the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has a high level of control (estimated to be 90% of the control) over the glycolytic flux in nonproliferating cells of Lactococcus lactis (33). However, it has recently been shown that GAPDH has no control over the glycolytic flux in steadily growing L. lactis cells (Solem, Koebmann, and Jensen, unpublished data). The control over the glycolytic flux exerted by lactate dehydrogenase was also reported to be close to zero (2).According to metabolic control theory (16, 25), flux control can reside in any of the steps in a system; i.e., it can reside in the numerous processes that consume the ATP generated in glycolysis (8,17). Indeed, we have recently shown that at least 75% of the control over glycolysis in aerobic Escherichia coli cultures occurs in the ATP-consuming reactions (26). This result was obtained by overexpression of genes encoding part of the F 1 unit of the (F 1 F 0 ) H ϩ -ATPase, which resulted in uncoupling of glycolysis from biomass production and a 70% increase in the glycolytic flux.In this paper we show that expression of genes encoding...

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Section: Discussionsupporting

confidence: 92%

Expression of Genes Encoding F ₁ -ATPase Results in Uncoupling of Glycolysis from Biomass Production in Lactococcus lactis

Koebmann

Solem

Pedersen³

et al. 2002

Appl Environ Microbiol

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“…Polyclonal Antibodies for Nha1p or Cos3p-His 6 -tagged Nha1p (C1ϩC2) (Nha1p (C1ϩC2)-His) bearing the Nha1p amino acid residues 434 -523 and the MBP-Cos3p-loop fusion bearing the Cos3p amino acid residues 94 -224 were overproduced in E. coli BL21 and purified by affinity chromatography with Ni-NTA-agarose (Qiagen) and glutathione-Sepharose beads (Amersham Biosciences), respectively (49). The purified Nha1p (C1ϩC2)-His and MBP-Cos3p-loop proteins were injected into rabbits to induce polyclonal antibodies.…”

Section: Analysis Of Protein-proteinmentioning

confidence: 99%

A Novel Membrane Protein Capable of Binding the Na+/H+ Antiporter (Nha1p) Enhances the Salinity-resistant Cell Growth of Saccharomyces cerevisiae

Mitsui

Ochi

Nakamura

et al. 2004

Journal of Biological Chemistry

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The Na؉ /H ؉ antiporter Nha1p of Saccharomyces cerevisiae plays an important role in maintaining intracellular pH and Na ؉ homeostasis. Nha1p has a two-domain structure composed of integral membrane and hydrophilic tail regions. Overexpression of a peptide of ϳ40 residues (C1؉C2 domains) that is localized in the juxtamembrane area of its cytoplasmic tail caused cell growth retardation in highly saline conditions, possibly by decreasing Na ؉ /H ؉ antiporter activity. A multicopy suppressor gene of this growth retardation was identified from a yeast genome library. The clone encodes a novel membrane protein denoted as COS3 in the genome data base. Overexpression or deletion of COS3 increases or decreases salinity-resistant cell growth, respectively. However, in nha1⌬ cells, overexpression of COS3 alone did not suppress the growth retardation. Cos3p and a hydrophilic portion of Cos3p interact with the C1؉C2 peptide in vitro, and Cos3p is co-precipitated with Nha1p from yeast cell extracts. Cos3p-GFP mainly resides at the vacuole, but overexpression of Nha1p caused a portion of the Cos3p-GFP proteins to shift to the cytoplasmic membrane. These observations suggest that Cos3p is a novel membrane protein that can enhance salinity-resistant cell growth by interacting with the C1؉C2 domain of Nha1p and thereby possibly activating the antiporter activity of this protein.

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“…His tags were added to β subunits in order to precisely position and orient F 1 -ATPase molecules on nanofabricated substrates. Previous research demonstrated a 40% reduction in enzymatic activity associated with the addition of His tags to the F 1 -ATPase from E. coli (Ekuni et al 1998). For this reason, the mutated F 1 -ATPase constructs with and without His tags on the β subunit are currently being cloned into pQE-30, and expressed in E. coli JM103.…”

Section: F 1 -Atpase Biomolecular Motor Productionmentioning

confidence: 99%

Constructing nanomechanical devices powered by biomolecular motors

1999

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A confluence in scientific advancements associated with molecular biology and nanofabrication technology now offers the potential of engineering functional hybrid organic/inorganic nanomechanical systems. Our objectives were to: (i) establish a system for producing a recombinant biomolecular motor; (ii) precisely position and orient biological molecules on nanofabricated substrates; and (iii) acquire baseline performance data on a biomolecular motor in a hybrid system. A recombinant expression system was established for the large-scale production of a thermostable biomolecular motor, F1-ATPase, modified to contain chemically active `handles.' His tags were used to specifically attach, as well as precisely position and orient, biological molecules on nickel, copper and gold substrates created using electron beam lithography. Further, these substrates were used to attach F1-ATPase and acquire baseline performance data on motor rotation through the attachment of fluorescent microspheres to the tip of the γ subunit. Counterclockwise rotation of the γ subunit was measured at approximately 10 Hz (3-4 rev s-1) using a differential interferometer. These data have established several prerequisite technologies that are essential to the integration of biomolecular motors in nano-electro-mechanical systems. The evolution of these technologies will open the door to the seamless integration of the motive power of life with engineered nanofabricated devices.

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Reconstitution of F₁‐ATPase activity from Escherichia coli subunits α, β and subunit γ tagged with six histidine residues at the C‐terminus

Abstract: An engineered Q

Cited by 6 publications

References 29 publications

Expression of Genes Encoding F ₁ -ATPase Results in Uncoupling of Glycolysis from Biomass Production in Lactococcus lactis

Expression of Genes Encoding F ₁ -ATPase Results in Uncoupling of Glycolysis from Biomass Production in Lactococcus lactis

A Novel Membrane Protein Capable of Binding the Na+/H+ Antiporter (Nha1p) Enhances the Salinity-resistant Cell Growth of Saccharomyces cerevisiae

Constructing nanomechanical devices powered by biomolecular motors

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Reconstitution of F1‐ATPase activity from Escherichia coli subunits α, β and subunit γ tagged with six histidine residues at the C‐terminus

Abstract: An engineered Q

Cited by 6 publications

References 29 publications

Expression of Genes Encoding F 1 -ATPase Results in Uncoupling of Glycolysis from Biomass Production in Lactococcus lactis

Expression of Genes Encoding F 1 -ATPase Results in Uncoupling of Glycolysis from Biomass Production in Lactococcus lactis

A Novel Membrane Protein Capable of Binding the Na+/H+ Antiporter (Nha1p) Enhances the Salinity-resistant Cell Growth of Saccharomyces cerevisiae

Constructing nanomechanical devices powered by biomolecular motors

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Reconstitution of F₁‐ATPase activity from Escherichia coli subunits α, β and subunit γ tagged with six histidine residues at the C‐terminus

Expression of Genes Encoding F ₁ -ATPase Results in Uncoupling of Glycolysis from Biomass Production in Lactococcus lactis

Expression of Genes Encoding F ₁ -ATPase Results in Uncoupling of Glycolysis from Biomass Production in Lactococcus lactis