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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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Sam3 overexpression sensitizes yeast to sinefungin inhibition. Wild-type SAM3 cells were transformed with 2 µ SAM3 or 2 µ plasmids and grown in His− 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 His− agar plate (‘no drug’) and on His− plates onto which 150 µl of a 1.6 µM or 3.1 µM sinefungin solution had been applied and spread. If the applied drug is evenly distributed through the depth of the agar plate, these doses correspond to net sinefungin concentrations of about 10 and 19 nM, respectively. The plates were photographed after incubation for 3 days at 30°C.
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Figure 6: Sam3 overexpression sensitizes yeast to sinefungin inhibition. Wild-type SAM3 cells were transformed with 2 µ SAM3 or 2 µ plasmids and grown in His− 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 His− agar plate (‘no drug’) and on His− plates onto which 150 µl of a 1.6 µM or 3.1 µM sinefungin solution had been applied and spread. If the applied drug is evenly distributed through the depth of the agar plate, these doses correspond to net sinefungin concentrations of about 10 and 19 nM, respectively. The plates were photographed after incubation for 3 days at 30°C.

Mentions: We reasoned that if loss of Sam3 transporter function results in sinefungin resistance, then overexpression of Sam3 might enhance the sinefungin sensitivity of S. cerevisiae. Previous studies had established that introducing SAM3 on a high copy plasmid into a wild-type SAM3 strain elicited a 3.5-fold increase in AdoMet uptake (16). We expected that SAM3 overexpression would also increase sinefungin bioavailability. Thus, we cloned the wild-type SAM3 gene into a high-copy 2 µ HIS3 plasmid and then introduced the 2 µ SAM3 plasmid into wild-type yeast, in parallel with the empty 2 µ vector. The transformants were tested for growth on His− plates with no added drug and His− plates that had been overlaid with sinefungin. Preliminary experiments established that growth of wild-type yeast cells was suppressed by applying 150 µl of a 25–50 µM solution of sinefungin to the minimal agar medium. Thus, we tested for the effects of Sam3 overexpression at lower doses of sinefungin (150 µl of a 1.6 µM or 3.1 µM sinefungin solution) and observed that increasing the copy number of the SAM3 gene sensitized the cells to inhibition of growth by a sinefungin dose that had little impact on cells that had only a single copy of SAM3 (Figure 6). Thus, Sam3 is a tunable determinant of sinefungin sensitivity and resistance.Figure 6.


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)

Sam3 overexpression sensitizes yeast to sinefungin inhibition. Wild-type SAM3 cells were transformed with 2 µ SAM3 or 2 µ plasmids and grown in His− 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 His− agar plate (‘no drug’) and on His− plates onto which 150 µl of a 1.6 µM or 3.1 µM sinefungin solution had been applied and spread. If the applied drug is evenly distributed through the depth of the agar plate, these doses correspond to net sinefungin concentrations of about 10 and 19 nM, respectively. The plates were photographed after incubation for 3 days at 30°C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Sam3 overexpression sensitizes yeast to sinefungin inhibition. Wild-type SAM3 cells were transformed with 2 µ SAM3 or 2 µ plasmids and grown in His− 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 His− agar plate (‘no drug’) and on His− plates onto which 150 µl of a 1.6 µM or 3.1 µM sinefungin solution had been applied and spread. If the applied drug is evenly distributed through the depth of the agar plate, these doses correspond to net sinefungin concentrations of about 10 and 19 nM, respectively. The plates were photographed after incubation for 3 days at 30°C.
Mentions: We reasoned that if loss of Sam3 transporter function results in sinefungin resistance, then overexpression of Sam3 might enhance the sinefungin sensitivity of S. cerevisiae. Previous studies had established that introducing SAM3 on a high copy plasmid into a wild-type SAM3 strain elicited a 3.5-fold increase in AdoMet uptake (16). We expected that SAM3 overexpression would also increase sinefungin bioavailability. Thus, we cloned the wild-type SAM3 gene into a high-copy 2 µ HIS3 plasmid and then introduced the 2 µ SAM3 plasmid into wild-type yeast, in parallel with the empty 2 µ vector. The transformants were tested for growth on His− plates with no added drug and His− plates that had been overlaid with sinefungin. Preliminary experiments established that growth of wild-type yeast cells was suppressed by applying 150 µl of a 25–50 µM solution of sinefungin to the minimal agar medium. Thus, we tested for the effects of Sam3 overexpression at lower doses of sinefungin (150 µl of a 1.6 µM or 3.1 µM sinefungin solution) and observed that increasing the copy number of the SAM3 gene sensitized the cells to inhibition of growth by a sinefungin dose that had little impact on cells that had only a single copy of SAM3 (Figure 6). Thus, Sam3 is a tunable determinant of sinefungin sensitivity and resistance.Figure 6.

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