Pdc2 coordinates expression of the THI regulon in the yeast Saccharomyces cerevisiae

Abstract

Coordination of gene expression in response to different metabolic signals is crucial for cellular homeostasis. In this work, we addressed the role of Pdc2 in the coordinated control of biosynthesis and demand of an essential metabolic cofactor, thiaminediphosphate (ThDP). The DNA binding protein Pdc2 was initially identified as a regulator of the genes PDC1 and PDC5, which encode isoforms of the glycolytic enzyme pyruvate decarboxylase (Pdc). The Pdc2 has also been implicated as a regulator of genes encoding enzymes in ThDP metabolism. The ThDP is the cofactor of Pdc. Using global and gene-specific expression analysis, we show that Pdc2 is required for the upregulation of all genes controlled by thiamine availability. The Pdc2 seems to act together with Thi2, a known transcriptional regulator of THI genes. The requirement for these two factors differs in a gene-specific manner. While the Thi2, in conjunction with Thi3, seems to control expression of THI genes with respect to thiamine availability, the Pdc2 may link the ThDP demand to carbon source availability. Interestingly, the enzymes Pdc1 and Pdc5 are enriched in the nucleus. Both are known to affect gene expression in an autoregulatory mechanism and expression of both is regulated by glucose and Pdc2, further pointing to a role of Pdc2 in coordinating different metabolic signals. Our analysis helps to further define the THI regulon and hence the spectrum of genes/proteins involved in the ThDP homeostasis. In particular, we identify novel proteins putatively involved in thiamine and/or ThDP transport across the plasma and the mitochondrial membrane. In conclusion, the THI regulon is the most interesting system to study principles of genes expression and metabolic coordination and deserves further attention.

Identification of Yeast and Human 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAr) Transporters

“…mine is replete), these transporters are most probably poorly expressed and marginally contribute to AICAr uptake. We thus tested a third transporter, named Nrt1, suspected to contribute to thiamine uptake (30) and also identified as sensitive to chloroquine (29), a drug that we have shown to inhibit AICAr uptake in yeast (Fig. 5D).…”

“…6A), thus suggesting that AICAr could be taken up by other means. We hence explored the possible involvement of Nrt1 (previously identified as Thi71), a third transporter structurally related to Thi7, capable of taking up thiamine with low affinity (30) and known to be sensitive to chloroquine (29). Nrt1 is the major transporter of NmR (31), a precursor of NAD(H).…”

Industrial fuel ethanol yeasts contain adaptive copy number changes in genes involved in vitamin B1 and B6 biosynthesis

“…However, it is well known that for S. cerevisiae, as well as for other members of the Saccharomyces sensu stricto complex, the presence of thiamin in the medium has a negative effect on the initial growth phase of the yeast cells, although it does not affect the final cell density (Minami et al 1982;Nakamura et al 1982;Kamihara and Nakamura 1984). This thiamin-induced growth inhibition has been extensively studied in yeast, and is a consequence of the repressing effect of this vitamin on PLP biosynthesis (Minami et al 1982;Nakamura et al 1982;Kamihara and Nakamura 1984;Hohmann and Meacock 1998;RodriguezNavarro et al 2002;Mojzita and Hohmann 2006;Nosaka 2006). Due to the resultant marked decrease in cellular vitamin B6 (e.g., pyridoxine) content, many metabolic pathways are repressed, including a pronounced lowering of d-aminolevulinate synthase activity and decreased synthesis of heme, with consequent lowering of respiration and a significant alteration in membrane lipid composition due to inhibition of sterol and unsaturated fatty acids biosynthesis (Minami et al 1982;Nakamura et al 1982;Kamihara and Nakamura 1984).…”

“…Aside from the importance of vitamin B6 itself for amino acid metabolism and many other biochemical pathways, the biologically active form of vitamin B6 (pyridoxal 59-phosphate, or PLP) serves another role in S. cerevisiae as the precursor of thiamin (vitamin B1) (Rodriguez-Navarro et al 2002;Nosaka 2006). Vitamin B1 forms the cofactor thiamin pyrophosphate, which is essential for sugar utilization by S. cerevisiae, in particular for sugar fermentation (Bataillon et al 1996;Hohmann and Meacock 1998;Mojzita and Hohmann 2006). Indeed, several reports have shown significant up-regulation of thiamin metabolism genes in fermenting yeast cells in various industrial settings including wine, sake, and bread dough (Rossignol et al 2003;Tanaka et al 2006;Wu et al 2006), and in yeast cells cultivated in sugarcane molasses (Shima et al 2005), indicating that there is a high demand for thiamin (and its precursors) under these industrial conditions.…”

The genes and enzymes involved in the biosynthesis of thiamin and thiamin diphosphate in yeasts

“…The control occurs at the transcriptional level, and TDP serves as an intracellular negative signal. To date, three positively acting proteins, products of the THI2 (PHO6), THI3 and PDC2 genes, were found to regulate the expression of thiamin biosynthesis and transport genes [2,24,25]. The THI2 protein contains an N-terminal DNA-binding domain typical for many yeast transcriptional factors.…”