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Characterization of the l-alanine exporter AlaE of Escherichia coli and its potential role in protecting cells from a toxic-level accumulation of l-alanine and its derivatives.

Kim S, Ihara K, Katsube S, Hori H, Ando T, Isogai E, Yoneyama H - Microbiologyopen (2015)

Bottom Line: When 200 mmol/L l-alanine-loaded inverted membrane vesicles prepared from MLA301ΔalaE/pAlaE were placed in a solution containing 200 mmol/L or 0.34 μmol/L l-alanine, energy-dependent [(3) H]l-alanine accumulation occurred under either condition.This energy-dependent uphill accumulation of [(3) H]l-alanine was strongly inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone but not by dicyclohexylcarbodiimide, suggesting that the AlaE-mediated l-alanine extrusion was driven by proton motive force.Based on these results, physiological roles of the l-alanine exporter are discussed.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1, Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai, 981-8555, Japan.

No MeSH data available.


Related in: MedlinePlus

Accumulation of [3H]l-alanine (60 Ci mmol−1) in the l-alanine-loaded inverted membrane vesicles under an uphill solute potential. Inverted membrane vesicles were prepared from MLA301ΔalaE/pAlaE and MLA301ΔalaE. The extravesicular l-alanine concentration for the transport assay was 0.34 μmol/L. Transport assays were performed without NADH (A) and with 2.5 mmol/L NADH (B). Dotted line in (B) shows AlaE-mediated l-alanine accumulation obtained by subtracting the values in MLA301ΔalaE from those in MLA301ΔalaE/pAlaE in the presence of NADH. Values presented are the means of three independent experiments with standard deviations. Symbols: ○, MLA301ΔalaE/pAlaE; ●, MLA301ΔalaE.
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fig05: Accumulation of [3H]l-alanine (60 Ci mmol−1) in the l-alanine-loaded inverted membrane vesicles under an uphill solute potential. Inverted membrane vesicles were prepared from MLA301ΔalaE/pAlaE and MLA301ΔalaE. The extravesicular l-alanine concentration for the transport assay was 0.34 μmol/L. Transport assays were performed without NADH (A) and with 2.5 mmol/L NADH (B). Dotted line in (B) shows AlaE-mediated l-alanine accumulation obtained by subtracting the values in MLA301ΔalaE from those in MLA301ΔalaE/pAlaE in the presence of NADH. Values presented are the means of three independent experiments with standard deviations. Symbols: ○, MLA301ΔalaE/pAlaE; ●, MLA301ΔalaE.

Mentions: We next determined the [3H]l-alanine accumulation in the 200 mmol/L l-alanine-loaded membrane vesicles in the presence of only 0.34 μmol/L extravesicular [3H]l-alanine, a setup for an outward l-alanine chemical potential. The membrane vesicles prepared from MLA301ΔalaE/pAlaE rapidly accumulated [3H]l-alanine, reaching 43.3 fmol (mg protein)−1 within 1 min in the presence of NADH, and the level of labeled l-alanine was steadily maintained for at least 10 min (Fig.5B). In contrast, only a trace amount of l-alanine accumulation was observed in the absence of the energy source (Fig.5A). The vesicles prepared from MLA301ΔalaE showed a transient increase in intravesicular l-alanine, reaching 28 fmol (mg protein)−1 in 1 min in the presence of NADH, and then declined to the basal level after 10 min (Fig.5B). This energy-dependent l-alanine accumulation is likely due to exporters other than AlaE as reported earlier (Hori et al. 2011b). Subtraction of the l-alanine accumulation in MLA301ΔalaE vesicles from that of MLA301ΔalaE/pAlaE showed the actual AlaE-catalyzed energy-dependent uphill movement of l-alanine (Fig.5B, dotted line). Taken together, these results unequivocally demonstrate that AlaE catalyzes the active export of l-alanine in an energy-dependent manner.


Characterization of the l-alanine exporter AlaE of Escherichia coli and its potential role in protecting cells from a toxic-level accumulation of l-alanine and its derivatives.

Kim S, Ihara K, Katsube S, Hori H, Ando T, Isogai E, Yoneyama H - Microbiologyopen (2015)

Accumulation of [3H]l-alanine (60 Ci mmol−1) in the l-alanine-loaded inverted membrane vesicles under an uphill solute potential. Inverted membrane vesicles were prepared from MLA301ΔalaE/pAlaE and MLA301ΔalaE. The extravesicular l-alanine concentration for the transport assay was 0.34 μmol/L. Transport assays were performed without NADH (A) and with 2.5 mmol/L NADH (B). Dotted line in (B) shows AlaE-mediated l-alanine accumulation obtained by subtracting the values in MLA301ΔalaE from those in MLA301ΔalaE/pAlaE in the presence of NADH. Values presented are the means of three independent experiments with standard deviations. Symbols: ○, MLA301ΔalaE/pAlaE; ●, MLA301ΔalaE.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4554458&req=5

fig05: Accumulation of [3H]l-alanine (60 Ci mmol−1) in the l-alanine-loaded inverted membrane vesicles under an uphill solute potential. Inverted membrane vesicles were prepared from MLA301ΔalaE/pAlaE and MLA301ΔalaE. The extravesicular l-alanine concentration for the transport assay was 0.34 μmol/L. Transport assays were performed without NADH (A) and with 2.5 mmol/L NADH (B). Dotted line in (B) shows AlaE-mediated l-alanine accumulation obtained by subtracting the values in MLA301ΔalaE from those in MLA301ΔalaE/pAlaE in the presence of NADH. Values presented are the means of three independent experiments with standard deviations. Symbols: ○, MLA301ΔalaE/pAlaE; ●, MLA301ΔalaE.
Mentions: We next determined the [3H]l-alanine accumulation in the 200 mmol/L l-alanine-loaded membrane vesicles in the presence of only 0.34 μmol/L extravesicular [3H]l-alanine, a setup for an outward l-alanine chemical potential. The membrane vesicles prepared from MLA301ΔalaE/pAlaE rapidly accumulated [3H]l-alanine, reaching 43.3 fmol (mg protein)−1 within 1 min in the presence of NADH, and the level of labeled l-alanine was steadily maintained for at least 10 min (Fig.5B). In contrast, only a trace amount of l-alanine accumulation was observed in the absence of the energy source (Fig.5A). The vesicles prepared from MLA301ΔalaE showed a transient increase in intravesicular l-alanine, reaching 28 fmol (mg protein)−1 in 1 min in the presence of NADH, and then declined to the basal level after 10 min (Fig.5B). This energy-dependent l-alanine accumulation is likely due to exporters other than AlaE as reported earlier (Hori et al. 2011b). Subtraction of the l-alanine accumulation in MLA301ΔalaE vesicles from that of MLA301ΔalaE/pAlaE showed the actual AlaE-catalyzed energy-dependent uphill movement of l-alanine (Fig.5B, dotted line). Taken together, these results unequivocally demonstrate that AlaE catalyzes the active export of l-alanine in an energy-dependent manner.

Bottom Line: When 200 mmol/L l-alanine-loaded inverted membrane vesicles prepared from MLA301ΔalaE/pAlaE were placed in a solution containing 200 mmol/L or 0.34 μmol/L l-alanine, energy-dependent [(3) H]l-alanine accumulation occurred under either condition.This energy-dependent uphill accumulation of [(3) H]l-alanine was strongly inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone but not by dicyclohexylcarbodiimide, suggesting that the AlaE-mediated l-alanine extrusion was driven by proton motive force.Based on these results, physiological roles of the l-alanine exporter are discussed.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1, Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai, 981-8555, Japan.

No MeSH data available.


Related in: MedlinePlus