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Sinefungin resistance of Saccharomyces cerevisiae arising from Sam3 mutations that inactivate the AdoMet transporter or from increased expression of AdoMet synthase plus mRNA cap guanine-N7 methyltransferase.

Zheng S, Shuman S, Schwer B - Nucleic Acids Res. (2007)

Bottom Line: Thus, Sam3 is a tunable determinant of sinefungin potency.Insights to the intracellular action of sinefungin stem from the finding that increased gene dosage of yeast AdoMet synthase plus cap guanine-N7 methyltransferase afforded greater resistance to sinefungin than either enzyme alone.These results are consistent with the proposal that mRNA cap methylation is a principal target of sinefungin's bioactivity.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Program, Sloan-Kettering Institute and Microbiology, Weill Cornell Medical College, New York, NY 10065, USA.

ABSTRACT
The S-adenosylmethionine (AdoMet) analog sinefungin is a natural product antibiotic that inhibits nucleic acid methyltransferases and arrests the growth of unicellular eukarya and eukaryal viruses. The basis for the particular sensitivity of fungi and protozoa to sinefungin is not known. Here we report the isolation and characterization of spontaneous sinefungin-resistant mutants of the budding yeast Saccharomyces cerevisiae. In all cases, sinefungin resistance was attributable to a loss-of-function mutation in Sam3, the yeast high-affinity AdoMet transporter. Overexpression of wild-type Sam3 increased the sensitivity of yeast to growth inhibition by sinefungin. Thus, Sam3 is a tunable determinant of sinefungin potency. The shared ability of protozoan parasites to import AdoMet might determine sinefungin's anti-infective spectrum. Insights to the intracellular action of sinefungin stem from the finding that increased gene dosage of yeast AdoMet synthase plus cap guanine-N7 methyltransferase afforded greater resistance to sinefungin than either enzyme alone. These results are consistent with the proposal that mRNA cap methylation is a principal target of sinefungin's bioactivity.

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Related in: MedlinePlus

The sfr strains are defective in utilization of exogenous AdoMet. The indicated yeast strains were grown in YPD medium at 30°C until A600 reached ∼0.7. The cells were harvested by centrifugation, suspended in water, recentrifuged and resuspended in water. Serial 10-fold dilutions (in water) of the washed cells were prepared and aliquots (2 µl) were spotted on B medium agarose plates or B medium supplemented with 0.5 mM AdoMet or 0.1 mM methionine. The plates were photographed after incubation for 2 days at 30°C.
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Figure 2: The sfr strains are defective in utilization of exogenous AdoMet. The indicated yeast strains were grown in YPD medium at 30°C until A600 reached ∼0.7. The cells were harvested by centrifugation, suspended in water, recentrifuged and resuspended in water. Serial 10-fold dilutions (in water) of the washed cells were prepared and aliquots (2 µl) were spotted on B medium agarose plates or B medium supplemented with 0.5 mM AdoMet or 0.1 mM methionine. The plates were photographed after incubation for 2 days at 30°C.

Mentions: To test whether the four sfr strains were defective in utilizing exogenous AdoMet, we spotted serial dilutions of wild-type, sfr, and sam3Δ cells on unsupplemented B medium and on B medium containing either AdoMet or methionine as the sulfur source (Figure 2). All of the strains were impaired for growth on B medium. The sam3Δ mutant was unable to grow using AdoMet as the sulfur source, yet it was able to grow in the presence of methionine, as reported previously (16). The wild-type strain was the only one for which growth was restored by AdoMet. Each of the sfr strains phenocopied sam3Δ with respect to their ability to utilize methionine but not AdoMet (Figure 2). These results underscore a correlation between impaired AdoMet uptake and sinefungin-resistance in yeast. Yet, they do not reveal whether the sfr mutations elicit a sam3-like phenotype directly, via mutations in Sam3, or indirectly, through mutations in genes that regulate Sam3 expression or function.Figure 2.


Sinefungin resistance of Saccharomyces cerevisiae arising from Sam3 mutations that inactivate the AdoMet transporter or from increased expression of AdoMet synthase plus mRNA cap guanine-N7 methyltransferase.

Zheng S, Shuman S, Schwer B - Nucleic Acids Res. (2007)

The sfr strains are defective in utilization of exogenous AdoMet. The indicated yeast strains were grown in YPD medium at 30°C until A600 reached ∼0.7. The cells were harvested by centrifugation, suspended in water, recentrifuged and resuspended in water. Serial 10-fold dilutions (in water) of the washed cells were prepared and aliquots (2 µl) were spotted on B medium agarose plates or B medium supplemented with 0.5 mM AdoMet or 0.1 mM methionine. The plates were photographed after incubation for 2 days at 30°C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175321&req=5

Figure 2: The sfr strains are defective in utilization of exogenous AdoMet. The indicated yeast strains were grown in YPD medium at 30°C until A600 reached ∼0.7. The cells were harvested by centrifugation, suspended in water, recentrifuged and resuspended in water. Serial 10-fold dilutions (in water) of the washed cells were prepared and aliquots (2 µl) were spotted on B medium agarose plates or B medium supplemented with 0.5 mM AdoMet or 0.1 mM methionine. The plates were photographed after incubation for 2 days at 30°C.
Mentions: To test whether the four sfr strains were defective in utilizing exogenous AdoMet, we spotted serial dilutions of wild-type, sfr, and sam3Δ cells on unsupplemented B medium and on B medium containing either AdoMet or methionine as the sulfur source (Figure 2). All of the strains were impaired for growth on B medium. The sam3Δ mutant was unable to grow using AdoMet as the sulfur source, yet it was able to grow in the presence of methionine, as reported previously (16). The wild-type strain was the only one for which growth was restored by AdoMet. Each of the sfr strains phenocopied sam3Δ with respect to their ability to utilize methionine but not AdoMet (Figure 2). These results underscore a correlation between impaired AdoMet uptake and sinefungin-resistance in yeast. Yet, they do not reveal whether the sfr mutations elicit a sam3-like phenotype directly, via mutations in Sam3, or indirectly, through mutations in genes that regulate Sam3 expression or function.Figure 2.

Bottom Line: Thus, Sam3 is a tunable determinant of sinefungin potency.Insights to the intracellular action of sinefungin stem from the finding that increased gene dosage of yeast AdoMet synthase plus cap guanine-N7 methyltransferase afforded greater resistance to sinefungin than either enzyme alone.These results are consistent with the proposal that mRNA cap methylation is a principal target of sinefungin's bioactivity.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Program, Sloan-Kettering Institute and Microbiology, Weill Cornell Medical College, New York, NY 10065, USA.

ABSTRACT
The S-adenosylmethionine (AdoMet) analog sinefungin is a natural product antibiotic that inhibits nucleic acid methyltransferases and arrests the growth of unicellular eukarya and eukaryal viruses. The basis for the particular sensitivity of fungi and protozoa to sinefungin is not known. Here we report the isolation and characterization of spontaneous sinefungin-resistant mutants of the budding yeast Saccharomyces cerevisiae. In all cases, sinefungin resistance was attributable to a loss-of-function mutation in Sam3, the yeast high-affinity AdoMet transporter. Overexpression of wild-type Sam3 increased the sensitivity of yeast to growth inhibition by sinefungin. Thus, Sam3 is a tunable determinant of sinefungin potency. The shared ability of protozoan parasites to import AdoMet might determine sinefungin's anti-infective spectrum. Insights to the intracellular action of sinefungin stem from the finding that increased gene dosage of yeast AdoMet synthase plus cap guanine-N7 methyltransferase afforded greater resistance to sinefungin than either enzyme alone. These results are consistent with the proposal that mRNA cap methylation is a principal target of sinefungin's bioactivity.

Show MeSH
Related in: MedlinePlus