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Identification of novel secreted fatty acids that regulate nitrogen catabolite repression in fission yeast.

Sun X, Hirai G, Ueki M, Hirota H, Wang Q, Hongo Y, Nakamura T, Hitora Y, Takahashi H, Sodeoka M, Osada H, Hamamoto M, Yoshida M, Yashiroda Y - Sci Rep (2016)

Bottom Line: Synthetic NSFs were also able to shift nitrogen source utilization from high-quality to poor nitrogen sources to allow adaptive growth of the fission yeast amino acid auxotrophic mutants in the presence of high-quality nitrogen sources.Finally, we demonstrated that the Agp3 amino acid transporter was involved in the adaptive growth.The data highlight a novel intra-species communication system for adaptation to environmental nutritional conditions in fission yeast.

View Article: PubMed Central - PubMed

Affiliation: Chemical Genetics Laboratory, RIKEN, Saitama, Japan.

ABSTRACT
Uptake of poor nitrogen sources such as branched-chain amino acids is repressed in the presence of high-quality nitrogen sources such as NH4(+) and glutamate (Glu), which is called nitrogen catabolite repression. Amino acid auxotrophic mutants of the fission yeast Schizosaccharomyces pombe were unable to grow on minimal medium containing NH4Cl or Glu even when adequate amounts of required amino acids were supplied. However, growth of these mutant cells was recovered in the vicinity of colonies of the prototrophic strain, suggesting that the prototrophic cells secrete some substances that can restore uptake of amino acids by an unknown mechanism. We identified the novel fatty acids, 10(R)-acetoxy-8(Z)-octadecenoic acid and 10(R)-hydroxy-8(Z)-octadecenoic acid, as secreted active substances, referred to as Nitrogen Signaling Factors (NSFs). Synthetic NSFs were also able to shift nitrogen source utilization from high-quality to poor nitrogen sources to allow adaptive growth of the fission yeast amino acid auxotrophic mutants in the presence of high-quality nitrogen sources. Finally, we demonstrated that the Agp3 amino acid transporter was involved in the adaptive growth. The data highlight a novel intra-species communication system for adaptation to environmental nutritional conditions in fission yeast.

No MeSH data available.


Related in: MedlinePlus

Adaptive growth of the Leu auxotrophic mutant is dependent on Agp3.(a) Activity of synthetic 10(R)-hydroxy-8(Z)-octadecenoic acid ((R)-2) on the leu1 mutant. (R)-2 was dissolved in 50% MeOH, and a 2-fold dilution series of the compound was prepared with a starting concentration of 2.5 × 104 ng mL−1 to examine its activity. Solvent (50% MeOH) was used as a negative control (NC). Dilutions and solvent were layered onto solid minimal media containing 374 mM NH4Cl (EMM [374-N]) supplemented with 2 mM of Leu. leu1 mutant cells (SpHT81) suspended in water at OD600 = 0.02 were spotted onto the solid media, and plates were incubated at 30 °C for 5 days. (b) Screening of amino acid transporter genes involved in adaptive growth. Amino acid transporter gene mutants (SpHT478-489, and 502) and their parental strain (SpHT227) were cultured in EMM containing 187 mM NH4Cl (EMM [187-N]) supplemented with 2 mM each of Ade, Ura, and Leu at 30 °C for 48 hours. Growth was monitored by measuring OD600 in the presence of DMSO or 0.1 mg mL−1 of the supernatant of the prototrophic cell culture (SpHT219) (sup). cat1Δ mutant and agp3Δ mutant are indicated by arrows. The experiment was performed twice, and a representative result is shown. (c) Activity of synthetic 10(R)-hydroxy-8(Z)-octadecenoic acid ((R)-2) on the leu1 agp3Δ mutant (XY-21) and the leu1 cat1Δ mutant (XY-23). The experiment was prepared as described in (a).
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f3: Adaptive growth of the Leu auxotrophic mutant is dependent on Agp3.(a) Activity of synthetic 10(R)-hydroxy-8(Z)-octadecenoic acid ((R)-2) on the leu1 mutant. (R)-2 was dissolved in 50% MeOH, and a 2-fold dilution series of the compound was prepared with a starting concentration of 2.5 × 104 ng mL−1 to examine its activity. Solvent (50% MeOH) was used as a negative control (NC). Dilutions and solvent were layered onto solid minimal media containing 374 mM NH4Cl (EMM [374-N]) supplemented with 2 mM of Leu. leu1 mutant cells (SpHT81) suspended in water at OD600 = 0.02 were spotted onto the solid media, and plates were incubated at 30 °C for 5 days. (b) Screening of amino acid transporter genes involved in adaptive growth. Amino acid transporter gene mutants (SpHT478-489, and 502) and their parental strain (SpHT227) were cultured in EMM containing 187 mM NH4Cl (EMM [187-N]) supplemented with 2 mM each of Ade, Ura, and Leu at 30 °C for 48 hours. Growth was monitored by measuring OD600 in the presence of DMSO or 0.1 mg mL−1 of the supernatant of the prototrophic cell culture (SpHT219) (sup). cat1Δ mutant and agp3Δ mutant are indicated by arrows. The experiment was performed twice, and a representative result is shown. (c) Activity of synthetic 10(R)-hydroxy-8(Z)-octadecenoic acid ((R)-2) on the leu1 agp3Δ mutant (XY-21) and the leu1 cat1Δ mutant (XY-23). The experiment was prepared as described in (a).

Mentions: Adaptive growth has been observed not only in the eca39Δ mutant, but also in the Leu auxotrophic mutant leu13. In EMM containing Leu and excess NH4Cl, growth of the leu1 mutant was inhibited, but it was restored by the addition of supernatant of the prototrophic strain (Fig. S8a,b). By contrast, the growth of ade6 and ura4 single mutants was not promoted by supernatant of the prototrophic strain, suggesting that Leu uptake, but not Ade or Ura uptake, was specifically repressed by excess NH4Cl (Fig. S8b). We confirmed that both synthetic (R)-2 and (R)-1 isolated from fraction 1 induced adaptive growth of not only the eca39Δ mutant, but also the leu1 mutant, at very low concentrations, 1.5 ng/mL and 3.1 ng/mL, respectively (Fig. 3a and Fig. S9a). Therefore, we refer to the compounds (R)-1 and (R)-2 as “Nitrogen Signaling Factors (NSFs)” that induce the adaptive uptake of branched-chain amino acids into fission yeast cells.


Identification of novel secreted fatty acids that regulate nitrogen catabolite repression in fission yeast.

Sun X, Hirai G, Ueki M, Hirota H, Wang Q, Hongo Y, Nakamura T, Hitora Y, Takahashi H, Sodeoka M, Osada H, Hamamoto M, Yoshida M, Yashiroda Y - Sci Rep (2016)

Adaptive growth of the Leu auxotrophic mutant is dependent on Agp3.(a) Activity of synthetic 10(R)-hydroxy-8(Z)-octadecenoic acid ((R)-2) on the leu1 mutant. (R)-2 was dissolved in 50% MeOH, and a 2-fold dilution series of the compound was prepared with a starting concentration of 2.5 × 104 ng mL−1 to examine its activity. Solvent (50% MeOH) was used as a negative control (NC). Dilutions and solvent were layered onto solid minimal media containing 374 mM NH4Cl (EMM [374-N]) supplemented with 2 mM of Leu. leu1 mutant cells (SpHT81) suspended in water at OD600 = 0.02 were spotted onto the solid media, and plates were incubated at 30 °C for 5 days. (b) Screening of amino acid transporter genes involved in adaptive growth. Amino acid transporter gene mutants (SpHT478-489, and 502) and their parental strain (SpHT227) were cultured in EMM containing 187 mM NH4Cl (EMM [187-N]) supplemented with 2 mM each of Ade, Ura, and Leu at 30 °C for 48 hours. Growth was monitored by measuring OD600 in the presence of DMSO or 0.1 mg mL−1 of the supernatant of the prototrophic cell culture (SpHT219) (sup). cat1Δ mutant and agp3Δ mutant are indicated by arrows. The experiment was performed twice, and a representative result is shown. (c) Activity of synthetic 10(R)-hydroxy-8(Z)-octadecenoic acid ((R)-2) on the leu1 agp3Δ mutant (XY-21) and the leu1 cat1Δ mutant (XY-23). The experiment was prepared as described in (a).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4759587&req=5

f3: Adaptive growth of the Leu auxotrophic mutant is dependent on Agp3.(a) Activity of synthetic 10(R)-hydroxy-8(Z)-octadecenoic acid ((R)-2) on the leu1 mutant. (R)-2 was dissolved in 50% MeOH, and a 2-fold dilution series of the compound was prepared with a starting concentration of 2.5 × 104 ng mL−1 to examine its activity. Solvent (50% MeOH) was used as a negative control (NC). Dilutions and solvent were layered onto solid minimal media containing 374 mM NH4Cl (EMM [374-N]) supplemented with 2 mM of Leu. leu1 mutant cells (SpHT81) suspended in water at OD600 = 0.02 were spotted onto the solid media, and plates were incubated at 30 °C for 5 days. (b) Screening of amino acid transporter genes involved in adaptive growth. Amino acid transporter gene mutants (SpHT478-489, and 502) and their parental strain (SpHT227) were cultured in EMM containing 187 mM NH4Cl (EMM [187-N]) supplemented with 2 mM each of Ade, Ura, and Leu at 30 °C for 48 hours. Growth was monitored by measuring OD600 in the presence of DMSO or 0.1 mg mL−1 of the supernatant of the prototrophic cell culture (SpHT219) (sup). cat1Δ mutant and agp3Δ mutant are indicated by arrows. The experiment was performed twice, and a representative result is shown. (c) Activity of synthetic 10(R)-hydroxy-8(Z)-octadecenoic acid ((R)-2) on the leu1 agp3Δ mutant (XY-21) and the leu1 cat1Δ mutant (XY-23). The experiment was prepared as described in (a).
Mentions: Adaptive growth has been observed not only in the eca39Δ mutant, but also in the Leu auxotrophic mutant leu13. In EMM containing Leu and excess NH4Cl, growth of the leu1 mutant was inhibited, but it was restored by the addition of supernatant of the prototrophic strain (Fig. S8a,b). By contrast, the growth of ade6 and ura4 single mutants was not promoted by supernatant of the prototrophic strain, suggesting that Leu uptake, but not Ade or Ura uptake, was specifically repressed by excess NH4Cl (Fig. S8b). We confirmed that both synthetic (R)-2 and (R)-1 isolated from fraction 1 induced adaptive growth of not only the eca39Δ mutant, but also the leu1 mutant, at very low concentrations, 1.5 ng/mL and 3.1 ng/mL, respectively (Fig. 3a and Fig. S9a). Therefore, we refer to the compounds (R)-1 and (R)-2 as “Nitrogen Signaling Factors (NSFs)” that induce the adaptive uptake of branched-chain amino acids into fission yeast cells.

Bottom Line: Synthetic NSFs were also able to shift nitrogen source utilization from high-quality to poor nitrogen sources to allow adaptive growth of the fission yeast amino acid auxotrophic mutants in the presence of high-quality nitrogen sources.Finally, we demonstrated that the Agp3 amino acid transporter was involved in the adaptive growth.The data highlight a novel intra-species communication system for adaptation to environmental nutritional conditions in fission yeast.

View Article: PubMed Central - PubMed

Affiliation: Chemical Genetics Laboratory, RIKEN, Saitama, Japan.

ABSTRACT
Uptake of poor nitrogen sources such as branched-chain amino acids is repressed in the presence of high-quality nitrogen sources such as NH4(+) and glutamate (Glu), which is called nitrogen catabolite repression. Amino acid auxotrophic mutants of the fission yeast Schizosaccharomyces pombe were unable to grow on minimal medium containing NH4Cl or Glu even when adequate amounts of required amino acids were supplied. However, growth of these mutant cells was recovered in the vicinity of colonies of the prototrophic strain, suggesting that the prototrophic cells secrete some substances that can restore uptake of amino acids by an unknown mechanism. We identified the novel fatty acids, 10(R)-acetoxy-8(Z)-octadecenoic acid and 10(R)-hydroxy-8(Z)-octadecenoic acid, as secreted active substances, referred to as Nitrogen Signaling Factors (NSFs). Synthetic NSFs were also able to shift nitrogen source utilization from high-quality to poor nitrogen sources to allow adaptive growth of the fission yeast amino acid auxotrophic mutants in the presence of high-quality nitrogen sources. Finally, we demonstrated that the Agp3 amino acid transporter was involved in the adaptive growth. The data highlight a novel intra-species communication system for adaptation to environmental nutritional conditions in fission yeast.

No MeSH data available.


Related in: MedlinePlus