Genetic analysis of the Warburg effect in yeast

Abstract

We recently discovered that the Warburg effect, defined by the dramatically enhanced metabolism of glucose to pyruvate, even in well-oxygenated cancer cells, can occur as a consequence of mutations that enhance lipid biosynthesis at the expense of respiratory capacity. Specifically, mutations in the E1 subunit of either of two respiratory enzymes, pyruvate dehydrogenase (PDC) or α-ketoglutarate dehydrogenase (KGDC), change substrate specificity from the 3-carbon α-ketoacid pyruvate, or the 5-carbon α-ketoacid α-ketoglutarate, to the 4-carbon α-ketoacid oxaloacetate (OADC). These mutations result in OADC-catalyzed synthesis of malonyl-CoA (MaCoA), the essential precursor of all fatty acids. These mutants arose as spontaneous suppressors of a yeast acc1(cs) cold-sensitive mutation encoding an altered form of AcCoA carboxylase (Acc1) that fails to produce MaCoA at the restrictive temperature (16 °C). Notably, these suppressors are respiratory defective as a result of the same nuclear mutations that suppress acc1(cs). These mutants also suppress sensitivity to Soraphen A, a potent inhibitor of Acc1 activity, at normal temperature (30 °C). To our knowledge, OADC activity has never been identified in eukaryotic cells. Our results offer a novel perspective on the Warburg effect: the reprogramming of energy metabolism in cancer cells as a consequence of mutational impairment of respiration to meet the fatty acid requirements of rapidly proliferating cells. We suggest OADC activity is a common feature of cancer cells and represents a novel target for the development of chemotherapeutics.

Keywords: AcCoA carboxylase; Cancer cell metabolism; Warburg effect; α-Ketoacid dehydrogenase.

Fig. 1

Growth phenotypes for the R64 suppressor of acc1^cs. The right panel depicts growth of strains on 2% glucose medium at 30°C (2 days); the middle panel depicts growth on 2% glucose at 16°C (10 days); the right panel depicts growth on 3% glycerol medium (3 days). The red lines divide diploid (2N) and haploid (1N) strains. Key: WT (ACC1⁺); H-470 (MATα acc1^cs); 1341 (MATa acc1^cs); R64 (acc1^cs pdb1^sup). Haploid strains show the tight Csm⁻ phenotype of acc1^cs, and the effective suppression of Csm⁻ by the R64 suppressor (middle panel). The R64 × 1341 backcross shows that the R64 suppressor is recessive with respect to Gly⁻, but dominant with respect to Csm⁺. The other three diploid strains show that the R64 Gly⁻ phenotype is not complemented when crossed with a pdb1Δ deletion strain, but is complemented with crossed with pda1Δ and kgd1Δ strains. This is compelling genetic evidence that the R64 suppressor is a pdb1 allele.

Fig. 2

Complementation of acc1^cs suppressor strains R34 and R64. Growth phenotypes of acc1^cs suppressor strains R34 and R64 into which a LEU2 vector alone (V) or the same vector carry the wild type PDA1 or PDB1 gene was introduced. Transformants were selected on –Leu, 2% glucose medium, then scored for respiratory competence on –Leu, 3% glycerol medium. Growth was photographed after two days incubation at 30°C.

Fig. 3

Suppression of Soraphen A toxicity. The acc1^cs mutant (H-470) is extremely sensitive to Soraphen A, a potent inhibitor of AcCoA carboxylase, even at the permissive temperature of 30°C. This growth defect is effectively suppressed by the acc1^cs suppressor strains R3, R34, R64 and, to a lesser extent, R92. The plate was photographed after 2 days incubation at 30°C. Note: The H-470 (acc1^cs) sensitivity to Soraphen A is at 30°C, identical to the Csm⁻ phenotype of H-470 in the absence of Soraphen A at 16°C (Fig. 1).