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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

Effect of CCCP and DCCD on the accumulation of [3H]l-alanine in the l-alanine-loaded inverted membrane vesicles under an uphill solute potential. [3H]l-alanine accumulation was determined after 5 min of incubation as described in Materials and Methods. The AlaE-mediated [3H]l-alanine (38 Ci mmol−1) accumulation was calculated by subtracting values obtained with MLA301ΔalaE vesicles from those in MLA301ΔalaE/pAlaE in the presence of NADH. Solid black bar, control; hatched bar, 20 μmol/L CCCP; solid gray bar, 50 μmol/L DCCD. Values presented are the means of three independent experiments with standard deviations (unpaired Student t-test, **<0.001).
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fig06: Effect of CCCP and DCCD on the accumulation of [3H]l-alanine in the l-alanine-loaded inverted membrane vesicles under an uphill solute potential. [3H]l-alanine accumulation was determined after 5 min of incubation as described in Materials and Methods. The AlaE-mediated [3H]l-alanine (38 Ci mmol−1) accumulation was calculated by subtracting values obtained with MLA301ΔalaE vesicles from those in MLA301ΔalaE/pAlaE in the presence of NADH. Solid black bar, control; hatched bar, 20 μmol/L CCCP; solid gray bar, 50 μmol/L DCCD. Values presented are the means of three independent experiments with standard deviations (unpaired Student t-test, **<0.001).

Mentions: To evaluate the nature of the energy source that drives the AlaE-mediated active export of l-alanine, we determined the [3H]l-alanine accumulation in the 200 mmol/L l-alanine-loaded membrane vesicles in the presence and absence of 20 μmol/L of carbonyl cyanide CCCP under an uphill solute gradient. As shown in Figure6, accumulation of [3H]l-alanine in the l-alanine-loaded MLA301ΔalaE/pAlaE vesicles decreased by approximately 74% after 5 min in the presence of CCCP compared to that obtained in the absence of CCCP. In contrast, when 50 μmol/L of N, N’-DCCD, an ATPase inhibitor (Hirata et al. 1974; Muller et al. 1987; Hermolin and Fillingame 1989), was present in the assay system, accumulation of [3H]l-alanine appeared comparable to that without inhibitor suggesting that ATP does not drive this active export system. An additional energy source conceivable may be the electrochemical potential of Na+ ion. However, this possibility appeared less likely as addressed in the discussion section. These results indicate that AlaE catalyzes the active export of l-alanine most likely utilizing proton electrochemical potential.


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)

Effect of CCCP and DCCD on the accumulation of [3H]l-alanine in the l-alanine-loaded inverted membrane vesicles under an uphill solute potential. [3H]l-alanine accumulation was determined after 5 min of incubation as described in Materials and Methods. The AlaE-mediated [3H]l-alanine (38 Ci mmol−1) accumulation was calculated by subtracting values obtained with MLA301ΔalaE vesicles from those in MLA301ΔalaE/pAlaE in the presence of NADH. Solid black bar, control; hatched bar, 20 μmol/L CCCP; solid gray bar, 50 μmol/L DCCD. Values presented are the means of three independent experiments with standard deviations (unpaired Student t-test, **<0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig06: Effect of CCCP and DCCD on the accumulation of [3H]l-alanine in the l-alanine-loaded inverted membrane vesicles under an uphill solute potential. [3H]l-alanine accumulation was determined after 5 min of incubation as described in Materials and Methods. The AlaE-mediated [3H]l-alanine (38 Ci mmol−1) accumulation was calculated by subtracting values obtained with MLA301ΔalaE vesicles from those in MLA301ΔalaE/pAlaE in the presence of NADH. Solid black bar, control; hatched bar, 20 μmol/L CCCP; solid gray bar, 50 μmol/L DCCD. Values presented are the means of three independent experiments with standard deviations (unpaired Student t-test, **<0.001).
Mentions: To evaluate the nature of the energy source that drives the AlaE-mediated active export of l-alanine, we determined the [3H]l-alanine accumulation in the 200 mmol/L l-alanine-loaded membrane vesicles in the presence and absence of 20 μmol/L of carbonyl cyanide CCCP under an uphill solute gradient. As shown in Figure6, accumulation of [3H]l-alanine in the l-alanine-loaded MLA301ΔalaE/pAlaE vesicles decreased by approximately 74% after 5 min in the presence of CCCP compared to that obtained in the absence of CCCP. In contrast, when 50 μmol/L of N, N’-DCCD, an ATPase inhibitor (Hirata et al. 1974; Muller et al. 1987; Hermolin and Fillingame 1989), was present in the assay system, accumulation of [3H]l-alanine appeared comparable to that without inhibitor suggesting that ATP does not drive this active export system. An additional energy source conceivable may be the electrochemical potential of Na+ ion. However, this possibility appeared less likely as addressed in the discussion section. These results indicate that AlaE catalyzes the active export of l-alanine most likely utilizing proton electrochemical potential.

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