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Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli.

Ohtsu I, Kawano Y, Suzuki M, Morigasaki S, Saiki K, Yamazaki S, Nonaka G, Takagi H - PLoS ONE (2015)

Bottom Line: Another protein YecSC is the FliY-dependent L-cystine transporter that functions cooperatively with the L-cystine transporter YdeD, which exports L-cystine as reducing equivalents from the cytoplasm to the periplasm, to prevent E. coli cells from oxidative stress.The exported L-cystine can reduce the periplasmic hydrogen peroxide to water, and then generated L-cystine is imported back into the cytoplasm via the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (Km = 110 nM) in a manner dependent on FliY, the periplasmic L-cystine-binding protein.From these findings, we propose that the hydrogen peroxide-inducible L-cystine/L-cystine shuttle system plays a role of detoxification of hydrogen peroxide before lipid peroxidation occurs, and then might specific prevent damage to membrane lipids.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan.

ABSTRACT
Intracellular thiols like L-cystine and L-cystine play a critical role in the regulation of cellular processes. Here we show that Escherichia coli has two L-cystine transporters, the symporter YdjN and the ATP-binding cassette importer FliY-YecSC. These proteins import L-cystine, an oxidized product of L-cystine from the periplasm to the cytoplasm. The symporter YdjN, which is expected to be a new member of the L-cystine regulon, is a low affinity L-cystine transporter (Km = 1.1 μM) that is mainly involved in L-cystine uptake from outside as a nutrient. E. coli has only two L-cystine importers because ΔydjNΔyecS mutant cells are not capable of growing in the minimal medium containing L-cystine as a sole sulfur source. Another protein YecSC is the FliY-dependent L-cystine transporter that functions cooperatively with the L-cystine transporter YdeD, which exports L-cystine as reducing equivalents from the cytoplasm to the periplasm, to prevent E. coli cells from oxidative stress. The exported L-cystine can reduce the periplasmic hydrogen peroxide to water, and then generated L-cystine is imported back into the cytoplasm via the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (Km = 110 nM) in a manner dependent on FliY, the periplasmic L-cystine-binding protein. The double disruption of ydeD and fliY increased cellular levels of lipid peroxides. From these findings, we propose that the hydrogen peroxide-inducible L-cystine/L-cystine shuttle system plays a role of detoxification of hydrogen peroxide before lipid peroxidation occurs, and then might specific prevent damage to membrane lipids.

No MeSH data available.


Related in: MedlinePlus

FliY-YecSC and YdjN are CySS importers with distinct properties.CySS uptake activity (initial rate) in the putative CySS importer gene disruptants (A) at various CySS concentrations and (B) in broader range of CySS concentrations to determine its kinetic parameters. Indicated concentrations of CySS containing radiolabeled [14C]-CySS was added to cells in assay buffer. After incubation for CySS uptake, cells were collected, and the amount of imported [14C]-CySS was detected by scintillation counter. (C) Hanes-Woolf plot of data shown in (B). (D) Induction of mRNA levels of CySS uptake and Cys transport genes by disruption of CySS importer gene. Cells of the exponential phase cultivated in LB medium (normal condition) were used for RT-PCR analysis. The catalase gene (katG) was also examined to judge the occurrence of oxidative stress by the gene disruption. The levels represent ratios compared to those of the same genes in wild-type cells.
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pone.0120619.g003: FliY-YecSC and YdjN are CySS importers with distinct properties.CySS uptake activity (initial rate) in the putative CySS importer gene disruptants (A) at various CySS concentrations and (B) in broader range of CySS concentrations to determine its kinetic parameters. Indicated concentrations of CySS containing radiolabeled [14C]-CySS was added to cells in assay buffer. After incubation for CySS uptake, cells were collected, and the amount of imported [14C]-CySS was detected by scintillation counter. (C) Hanes-Woolf plot of data shown in (B). (D) Induction of mRNA levels of CySS uptake and Cys transport genes by disruption of CySS importer gene. Cells of the exponential phase cultivated in LB medium (normal condition) were used for RT-PCR analysis. The catalase gene (katG) was also examined to judge the occurrence of oxidative stress by the gene disruption. The levels represent ratios compared to those of the same genes in wild-type cells.

Mentions: To examine whether FliY-YecSC and YdjN are the CySS importer, we measured the uptake rate of l-[14C]-CySS in the ΔfliY, ΔyecS, ΔyecC, ΔydjN, ΔydjNΔfliY, ΔydjNΔyecS, and ΔydjNΔyecC mutants at concentrations of 3.3–1,200 nM (Fig. 3A). As CySS concentration increased, the uptake rate of CySS in wild-type strain also increased, and reached ~15 pmol (109 cells)–1 min–1 at 1,200 nM CySS. In the ΔfliY, ΔyecS, ΔyecC, or ΔydjN mutants, the uptake rate significantly decreased to 45, 39, 37, or 58% of that in wild-type strain, respectively, at 1,200 nM CySS. Of the mutants, ΔfliY, ΔyecS, and ΔyecC displayed the same manner of CySS import, although only ΔydjN mutant showed a different pattern. Moreover, three types of double disruption, ydjN plus fliY, yecS, or yecC, abolished the CySS import. These results suggest that FliY, YecS, and YecC function in the same module as the CySS importer of FliY-YecSC, and that E. coli conclusively possesses only two CySS importers, FliY-YecSC and YdjN.


Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli.

Ohtsu I, Kawano Y, Suzuki M, Morigasaki S, Saiki K, Yamazaki S, Nonaka G, Takagi H - PLoS ONE (2015)

FliY-YecSC and YdjN are CySS importers with distinct properties.CySS uptake activity (initial rate) in the putative CySS importer gene disruptants (A) at various CySS concentrations and (B) in broader range of CySS concentrations to determine its kinetic parameters. Indicated concentrations of CySS containing radiolabeled [14C]-CySS was added to cells in assay buffer. After incubation for CySS uptake, cells were collected, and the amount of imported [14C]-CySS was detected by scintillation counter. (C) Hanes-Woolf plot of data shown in (B). (D) Induction of mRNA levels of CySS uptake and Cys transport genes by disruption of CySS importer gene. Cells of the exponential phase cultivated in LB medium (normal condition) were used for RT-PCR analysis. The catalase gene (katG) was also examined to judge the occurrence of oxidative stress by the gene disruption. The levels represent ratios compared to those of the same genes in wild-type cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4383340&req=5

pone.0120619.g003: FliY-YecSC and YdjN are CySS importers with distinct properties.CySS uptake activity (initial rate) in the putative CySS importer gene disruptants (A) at various CySS concentrations and (B) in broader range of CySS concentrations to determine its kinetic parameters. Indicated concentrations of CySS containing radiolabeled [14C]-CySS was added to cells in assay buffer. After incubation for CySS uptake, cells were collected, and the amount of imported [14C]-CySS was detected by scintillation counter. (C) Hanes-Woolf plot of data shown in (B). (D) Induction of mRNA levels of CySS uptake and Cys transport genes by disruption of CySS importer gene. Cells of the exponential phase cultivated in LB medium (normal condition) were used for RT-PCR analysis. The catalase gene (katG) was also examined to judge the occurrence of oxidative stress by the gene disruption. The levels represent ratios compared to those of the same genes in wild-type cells.
Mentions: To examine whether FliY-YecSC and YdjN are the CySS importer, we measured the uptake rate of l-[14C]-CySS in the ΔfliY, ΔyecS, ΔyecC, ΔydjN, ΔydjNΔfliY, ΔydjNΔyecS, and ΔydjNΔyecC mutants at concentrations of 3.3–1,200 nM (Fig. 3A). As CySS concentration increased, the uptake rate of CySS in wild-type strain also increased, and reached ~15 pmol (109 cells)–1 min–1 at 1,200 nM CySS. In the ΔfliY, ΔyecS, ΔyecC, or ΔydjN mutants, the uptake rate significantly decreased to 45, 39, 37, or 58% of that in wild-type strain, respectively, at 1,200 nM CySS. Of the mutants, ΔfliY, ΔyecS, and ΔyecC displayed the same manner of CySS import, although only ΔydjN mutant showed a different pattern. Moreover, three types of double disruption, ydjN plus fliY, yecS, or yecC, abolished the CySS import. These results suggest that FliY, YecS, and YecC function in the same module as the CySS importer of FliY-YecSC, and that E. coli conclusively possesses only two CySS importers, FliY-YecSC and YdjN.

Bottom Line: Another protein YecSC is the FliY-dependent L-cystine transporter that functions cooperatively with the L-cystine transporter YdeD, which exports L-cystine as reducing equivalents from the cytoplasm to the periplasm, to prevent E. coli cells from oxidative stress.The exported L-cystine can reduce the periplasmic hydrogen peroxide to water, and then generated L-cystine is imported back into the cytoplasm via the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (Km = 110 nM) in a manner dependent on FliY, the periplasmic L-cystine-binding protein.From these findings, we propose that the hydrogen peroxide-inducible L-cystine/L-cystine shuttle system plays a role of detoxification of hydrogen peroxide before lipid peroxidation occurs, and then might specific prevent damage to membrane lipids.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan.

ABSTRACT
Intracellular thiols like L-cystine and L-cystine play a critical role in the regulation of cellular processes. Here we show that Escherichia coli has two L-cystine transporters, the symporter YdjN and the ATP-binding cassette importer FliY-YecSC. These proteins import L-cystine, an oxidized product of L-cystine from the periplasm to the cytoplasm. The symporter YdjN, which is expected to be a new member of the L-cystine regulon, is a low affinity L-cystine transporter (Km = 1.1 μM) that is mainly involved in L-cystine uptake from outside as a nutrient. E. coli has only two L-cystine importers because ΔydjNΔyecS mutant cells are not capable of growing in the minimal medium containing L-cystine as a sole sulfur source. Another protein YecSC is the FliY-dependent L-cystine transporter that functions cooperatively with the L-cystine transporter YdeD, which exports L-cystine as reducing equivalents from the cytoplasm to the periplasm, to prevent E. coli cells from oxidative stress. The exported L-cystine can reduce the periplasmic hydrogen peroxide to water, and then generated L-cystine is imported back into the cytoplasm via the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (Km = 110 nM) in a manner dependent on FliY, the periplasmic L-cystine-binding protein. The double disruption of ydeD and fliY increased cellular levels of lipid peroxides. From these findings, we propose that the hydrogen peroxide-inducible L-cystine/L-cystine shuttle system plays a role of detoxification of hydrogen peroxide before lipid peroxidation occurs, and then might specific prevent damage to membrane lipids.

No MeSH data available.


Related in: MedlinePlus