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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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Increased gene dosage of SAM1 or SAM2 plus ABD1 confers sinefungin-resistance. (A) Yeast abd1Δ cells containing either a CEN TRP1 ABD1 plasmid (with ABD1 expression controlled by its native promoter) or a 2 µ TRP1 ABD1 plasmid (with ABD1 driven by the strong TPI1 promoter) were transformed with a 2 µ URA3 plasmid bearing either SAM1 or SAM2 or no insert. Transformants were grown in Ura− medium at 30°C until A600 reached ∼0.7. The cells were harvested by centrifugation and suspended in water. Serial 10-fold dilutions were prepared and aliquots (2 µl) were spotted on an unsupplemented Ura− agar plate (‘no drug’) and on Ura− plates onto which 150 µl of a 25, 50 or 100 µM sinefungin solution had been applied. If the applied drug is evenly distributed through the depth of the agar plate, these doses correspond to net sinefungin concentrations of 0.15, 0.3 and 0.6 µM, respectively. The plates were photographed after incubation for 3 days at 30°C. (B) Aliquots (2 µl) of serial 10-fold dilutions of wild-type yeast cells carrying either an empty CEN TRP1 vector or a 2 µ TRP1 ABD1 plasmid were spotted on an unsupplemented Trp− agar plate (‘no drug’) and on Trp− plates onto which 150 µl of a 25 µM sinefungin solution had been applied. The no drug and 25 µM sinefungin plates were photographed after incubation at 30°C for 3 days and 5 days, respectively.
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Figure 7: Increased gene dosage of SAM1 or SAM2 plus ABD1 confers sinefungin-resistance. (A) Yeast abd1Δ cells containing either a CEN TRP1 ABD1 plasmid (with ABD1 expression controlled by its native promoter) or a 2 µ TRP1 ABD1 plasmid (with ABD1 driven by the strong TPI1 promoter) were transformed with a 2 µ URA3 plasmid bearing either SAM1 or SAM2 or no insert. Transformants were grown in Ura− medium at 30°C until A600 reached ∼0.7. The cells were harvested by centrifugation and suspended in water. Serial 10-fold dilutions were prepared and aliquots (2 µl) were spotted on an unsupplemented Ura− agar plate (‘no drug’) and on Ura− plates onto which 150 µl of a 25, 50 or 100 µM sinefungin solution had been applied. If the applied drug is evenly distributed through the depth of the agar plate, these doses correspond to net sinefungin concentrations of 0.15, 0.3 and 0.6 µM, respectively. The plates were photographed after incubation for 3 days at 30°C. (B) Aliquots (2 µl) of serial 10-fold dilutions of wild-type yeast cells carrying either an empty CEN TRP1 vector or a 2 µ TRP1 ABD1 plasmid were spotted on an unsupplemented Trp− agar plate (‘no drug’) and on Trp− plates onto which 150 µl of a 25 µM sinefungin solution had been applied. The no drug and 25 µM sinefungin plates were photographed after incubation at 30°C for 3 days and 5 days, respectively.

Mentions: The genetic studies presented above highlight Sam3 as the major determinant of sinefungin susceptibility of budding yeast, but they do not illuminate the intracellular actions of the drug once it is taken up from the medium. If sinefungin is blocking growth by acting as a competitive inhibitor of AdoMet binding to an essential yeast methyltransferase, then we reasoned that increasing the intracellular level of AdoMet might ameliorate the effects of sinefungin on its methyltransferase target. S. cerevisiae encodes two AdoMet synthases: Sam1 and Sam2 (24). Previous findings that increased SAM1 or SAM2 gene dosage can suppress temperature-sensitive abd1 mutations (22) implied that Abd1 function is responsive to intracellular AdoMet levels. Here we introduced into yeast a multicopy 2 µ URA3 plasmid containing SAM1 or SAM2 under the control of the constitutive yeast TPI1 promoter (22). Compared to an empty 2 µ URA3 vector control, overexpression of either isozyme of AdoMet synthase enhanced cell growth on minimal medium overlaid with 150 µl of 25 µM or 50 µM sinefungin, but did not allow for growth at a higher drug dosage (Figure 7A). Transformation of yeast with a 2 µ TRP1 plasmid containing wild-type ABD1 driven by the TPI1 promoter also enabled growth on minimal medium dosed with 150 µl of 25 µM sinefungin (Figure 7B), but not with ≥50 µM sinefungin (not shown). Cotransformation with 2 µ ABD1 plus either 2 µ SAM1 or 2 µ SAM2 plasmids had an additive effect on sinefungin-resistance, whereby the strains overexpressing AdoMet synthase and cap methyltransferase grew on minimal medium overlaid with 150 µl of 100 µM sinefungin, a condition in which neither wild-type yeast nor yeast overexpressing Sam1 or Sam2 alone were able to form colonies (Figure 7A).Figure 7.


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)

Increased gene dosage of SAM1 or SAM2 plus ABD1 confers sinefungin-resistance. (A) Yeast abd1Δ cells containing either a CEN TRP1 ABD1 plasmid (with ABD1 expression controlled by its native promoter) or a 2 µ TRP1 ABD1 plasmid (with ABD1 driven by the strong TPI1 promoter) were transformed with a 2 µ URA3 plasmid bearing either SAM1 or SAM2 or no insert. Transformants were grown in Ura− medium at 30°C until A600 reached ∼0.7. The cells were harvested by centrifugation and suspended in water. Serial 10-fold dilutions were prepared and aliquots (2 µl) were spotted on an unsupplemented Ura− agar plate (‘no drug’) and on Ura− plates onto which 150 µl of a 25, 50 or 100 µM sinefungin solution had been applied. If the applied drug is evenly distributed through the depth of the agar plate, these doses correspond to net sinefungin concentrations of 0.15, 0.3 and 0.6 µM, respectively. The plates were photographed after incubation for 3 days at 30°C. (B) Aliquots (2 µl) of serial 10-fold dilutions of wild-type yeast cells carrying either an empty CEN TRP1 vector or a 2 µ TRP1 ABD1 plasmid were spotted on an unsupplemented Trp− agar plate (‘no drug’) and on Trp− plates onto which 150 µl of a 25 µM sinefungin solution had been applied. The no drug and 25 µM sinefungin plates were photographed after incubation at 30°C for 3 days and 5 days, respectively.
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Related In: Results  -  Collection

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Figure 7: Increased gene dosage of SAM1 or SAM2 plus ABD1 confers sinefungin-resistance. (A) Yeast abd1Δ cells containing either a CEN TRP1 ABD1 plasmid (with ABD1 expression controlled by its native promoter) or a 2 µ TRP1 ABD1 plasmid (with ABD1 driven by the strong TPI1 promoter) were transformed with a 2 µ URA3 plasmid bearing either SAM1 or SAM2 or no insert. Transformants were grown in Ura− medium at 30°C until A600 reached ∼0.7. The cells were harvested by centrifugation and suspended in water. Serial 10-fold dilutions were prepared and aliquots (2 µl) were spotted on an unsupplemented Ura− agar plate (‘no drug’) and on Ura− plates onto which 150 µl of a 25, 50 or 100 µM sinefungin solution had been applied. If the applied drug is evenly distributed through the depth of the agar plate, these doses correspond to net sinefungin concentrations of 0.15, 0.3 and 0.6 µM, respectively. The plates were photographed after incubation for 3 days at 30°C. (B) Aliquots (2 µl) of serial 10-fold dilutions of wild-type yeast cells carrying either an empty CEN TRP1 vector or a 2 µ TRP1 ABD1 plasmid were spotted on an unsupplemented Trp− agar plate (‘no drug’) and on Trp− plates onto which 150 µl of a 25 µM sinefungin solution had been applied. The no drug and 25 µM sinefungin plates were photographed after incubation at 30°C for 3 days and 5 days, respectively.
Mentions: The genetic studies presented above highlight Sam3 as the major determinant of sinefungin susceptibility of budding yeast, but they do not illuminate the intracellular actions of the drug once it is taken up from the medium. If sinefungin is blocking growth by acting as a competitive inhibitor of AdoMet binding to an essential yeast methyltransferase, then we reasoned that increasing the intracellular level of AdoMet might ameliorate the effects of sinefungin on its methyltransferase target. S. cerevisiae encodes two AdoMet synthases: Sam1 and Sam2 (24). Previous findings that increased SAM1 or SAM2 gene dosage can suppress temperature-sensitive abd1 mutations (22) implied that Abd1 function is responsive to intracellular AdoMet levels. Here we introduced into yeast a multicopy 2 µ URA3 plasmid containing SAM1 or SAM2 under the control of the constitutive yeast TPI1 promoter (22). Compared to an empty 2 µ URA3 vector control, overexpression of either isozyme of AdoMet synthase enhanced cell growth on minimal medium overlaid with 150 µl of 25 µM or 50 µM sinefungin, but did not allow for growth at a higher drug dosage (Figure 7A). Transformation of yeast with a 2 µ TRP1 plasmid containing wild-type ABD1 driven by the TPI1 promoter also enabled growth on minimal medium dosed with 150 µl of 25 µM sinefungin (Figure 7B), but not with ≥50 µM sinefungin (not shown). Cotransformation with 2 µ ABD1 plus either 2 µ SAM1 or 2 µ SAM2 plasmids had an additive effect on sinefungin-resistance, whereby the strains overexpressing AdoMet synthase and cap methyltransferase grew on minimal medium overlaid with 150 µl of 100 µM sinefungin, a condition in which neither wild-type yeast nor yeast overexpressing Sam1 or Sam2 alone were able to form colonies (Figure 7A).Figure 7.

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