Revisiting purine-histidine cross-pathway regulation in Saccharomyces cerevisiae: a central role for a small molecule

Abstract

Because some metabolic intermediates are involved in more than one pathway, crosstalk between pathways is crucial to maintaining homeostasis. AMP and histidine biosynthesis pathways are coregulated at the transcriptional level in response to adenine availability. 5'-Phosphoribosyl-4-carboxamide-5-aminoimidazole (AICAR), a metabolic intermediate at the crossroads between these two pathways, is shown here to be critical for activation of the transcriptional response in the absence of adenine. In this study, we show that both AMP and histidine pathways significantly contribute to AICAR synthesis. Furthermore, we show that upregulation of the histidine pathway clearly interferes with regulation of the AMP pathway, thus providing an explanation for the regulatory crosstalk between these pathways. Finally, we revisit the histidine auxotrophy of ade3 or ade16 ade17 mutants. Interestingly, overexpression of PMU1, encoding a potential phosphomutase, partially suppresses the histidine requirement of an ade3 ade16 ade17 triple mutant, most probably by reducing the level of AICAR in this mutant. Together our data clearly establish that AICAR is not just a metabolic intermediate but also acts as a true regulatory molecule.

Figure 1.—

Schematic of histidine and IMP biosynthesis pathways and reactions supplying 10 formyl-THF for IMP biosynthesis. FGAR, 5′-phosphoribosyl N-formylglycinamide; IMP, inosine 5′-monophosphate; PRPP, 5-phosphoribosyl-1-pyrophosphate; SAMP, adenylosuccinate; XMP, xanthosine 5′-monophosphate. Gene names are italicized. For simplicity, only intermediate metabolites that are cited in the text are indicated.

Figure 2.—

Effect of a mutation blocking IMP biosynthesis pathway on accumulation of AICAR. Yeast strains transformed with a plasmid carrying the ADE1-lacZ fusion (P115) were grown for 6 hr in SD casa medium containing adenine at either low (0.025 mm low ade) or high concentration (0.3 mm, high ade). β-Galactosidase activity was then measured as described in materials and methods. Strains were BY4742 (wild type), Y14244 (ade8), Y1095 (ade16 ade17), Y1164 (ade8 ade16 ade17), and Y16591 (ade3).

Figure 3.—

Expression of ADE1 and ADE17 in ade3 ade2 ade13 triple-mutant strains. Strains Y16591 (ade3) and Y1261 (ade2 ade13) were mated and after sporulation of the resulting diploid, tetrads were isolated. (A and B) The parental strains and the spores of two tetrads were transformed with a plasmid carrying the ADE1-lacZ fusion (P115) and grown for 6 hr in SD casa medium containing adenine at either low (0.025 mm) or high (0.3 mm) concentration. β-Gal activity was then measured as described in materials and methods. Strains were (A) parental strains Y1261 (ade2 ade13) and Y16591 (ade3); and (B) meiotic progeny strains Y1975 (wild type), Y1976 (ade2 ade3), Y1977 (ade2 ade3 ade13), Y1978 (ade13), Y1979 (ade2), Y1980 (ade3 ade13), Y1981 (ade2 ade3 ade13), and Y1982 (wild type). (C) Northern blot analysis of ADE17 gene expression in the ade2 ade3 ade13 mutant strain. Strains Y1975 (wild type), Y1261 (ade2 ade13), Y1980 (ade3 ade13), Y1976 (ade2 ade3), and Y1977 (ade2 ade3 ade13) were grown to an OD₆₀₀ of 0.5 in SD casa medium with or without adenine as indicated. Extraction of RNA and hybridization were performed as described in materials and methods. Radioactivity was detected using a Phosphorimager and signal was quantified using the ImageQuant software. The quantification is presented as the ADE17/ACT1 ratio, which was arbitrarily set up as “1” in the wild-type “+ade” control lane.

Figure 4.—

Effect of mutations blocking both the IMP and the histidine biosynthesis pathways from accumulating AICAR. (A–C). Yeast strains transformed with a plasmid carrying the ADE1-lacZ fusion (P115) were grown for 6 hr in SD casa medium containing adenine at either low (0.025 mm) or high (0.3 mm) concentration. β-Gal activity was then measured as described in materials and methods. Strains were: (A) BY4742 (wild type), Y16591 (ade3), Y10190 (his1), Y13388 (his7) Y1166 (ade3 his1), and Y1661 (ade3 his7); (B) BY4742 (wild type), Y16591 (ade3), Y1551 (ade3 ade5,7), and Y1554 (ade3 ade6); and (C) BY4742 (wild type), Y16591 (ade3), Y1166 (ade3 his1), Y1551 (ade3 ade5,7), and Y1657 (ade3 ade5,7 his1). (D) Northern blot analysis of ADE17 gene expression in the ade2 ade3 ade13 mutant strain. Strains BY4742 (wild type), Y10190 (his1), Y14601 (ade5,7), Y16591 (ade3), Y1166 (ade3 his1), Y1551 (ade3 ade5,7), and Y1657 (ade3 ade5,7 his1) were grown to an OD₆₀₀ of 0.5 in SD casa medium with or without adenine as indicated. Extraction of RNA and hybridization were performed as described in materials and methods. Radioactivity was detected using a phosphorimager and signal was quantified using the ImageQuant software. The quantification is presented as the ADE17/ACT1 ratio, which was arbitrarily set up as “1” in the wild-type “+ade” control lane.

Figure 5.—

Schematic of connections among histidine, purine, folate, and methionine metabolism in yeast. Gene names are italicized. CH + THF, methenyl-tetrahydrofolate; 5-CHO-THF, 5-formyl-tetrahydrofolate; CH₂-THF, methylene-tetrahydrofolate; CH₃-THF, methyl-tetrahydrofolate.

Figure 6.—

Phenotypes of double mutants combining ade3 mutations with mutations in folate metabolism. (A) Growth of the four spores of a tetratype obtained by mating Y06591 (ade3) and Y16179 (fau1) strains. A serial dilution of the spores was dropped onto YPD medium, and growth of cells was observed after 2 days at 30°. (B) Growth of the ade3 fau1 double mutant in the presence or the absence of methionine. Strains Y16591 (ade3), Y2030 (ade3 met6), Y2031 (ade3 fau1), and Y2032 (ade3 fau1) were streaked onto SD medium with (+met) or without (−met) methionine, and growth of the strains was observed after 3 days at 25° or 30°, as indicated. (C) The Y06591 (ade3) strain was mated with either Y13403 (shm1) or Y12669 (shm2) strains, and after sporulation of the resulting diploids, 12 tetrads (shown as vertical alignments of four spores) were dissected onto YPD medium supplemented with adenine (0.3 mm). Growth of spores was observed after 3 days at 30°.

Figure 7.—

Histidine auxotrophy of an ade3 ade16 ade17 triple mutant is suppressed by overexpression of PMU1. (A) A serial dilution of Y2406 transformed with P2818 (PMU1 2μ) or by the control vector was dropped onto SC medium with or without histidine as indicated. Pictures were taken after 3 days at 30° for the +histidine plate and 7 days for the −histidine plate. (B) Interruption of the PMU1 reading frame (P2819) abolishes suppression while overexpression of PMU1 alone (tet-PMU1) allows suppression. Suppression, indicated on the right, was monitored after transformation in the Y2406 strain. (C) Effect of accumulation of AICAR on cell growth. Strains were transformed with either a control plasmid (pCM189, vector) or a plasmid expressing the tet-ADE4 fusion (P1933, tet-ADE4). A serial dilution of the different transformants was dropped onto SD casa medium supplemented with adenine and tryptophan, and growth of cells was observed after 3 days at 30°. The following strains were used: Y1095 (ade16 ade17), BY4742 (wild type), Y11583 (ade16), Y16561 (ade17), Y16591 (ade3), and Y1164 (ade3 ade16 ade17). (D) Effect of overexpression of PMU1 on AICAR toxicity. A serial dilution of the Y1095 (ade16 ade17) strain transformed with tet-ADE4 (P2122) and either PMU1 overexpressing plasmids or the control vector was dropped onto SC medium with or without tetracycline as indicated. Growth of cells was observed after 3 days at 30°. (E) Toxicity of SAICAR accumulated in ade13 mutant strains. The following strains were used: PLY122 (wild type), 206 (ade13-52), 211 (ade13-23), and 242 (ade13-42). Residual adenylosuccinate lyase activity was measured in the four strains as described in materials and methods and indicated as a percentage of wild-type activity. Growth of cells was observed after 3 days at 30°.