In Saccharomyces cerevisiae, AMP biosynthesis genes (ADE genes) are transcriptionally activated in the absence of extracellular purines by the Bas1p and Bas2p (Pho2p) transcription factors. We now show that expression of the ADE genes is low in mutant strains affected in the first seven steps of the pathway, while it is constitutively derepressed in mutant strains affected in later steps. Combined with epistasy studies, these results show that 5-phosphoribosyl-4-succinocarboxamide-5-aminoimidazole (SAICAR), an intermediate metabolite of the pathway, is needed for optimal activation of the ADE genes. Two-hybrid studies establish that SAICAR is required to promote interaction between Bas1p and Bas2p in vivo, while in vitro experiments suggest that the effect of SAICAR on Bas1p-Bas2p interaction could be indirect. Importantly, feedback inhibition by ATP of Ade4p, catalyzing the first step of the pathway, appears to regulate SAICAR synthesis in response to adenine availability. Consistently, both ADE4 dominant mutations and overexpression of wild-type ADE4 lead to deregulation of ADE gene expression. We conclude that efficient transcription of yeast AMP biosynthesis genes requires interaction between Bas1p and Bas2p which is promoted in the presence of a metabolic intermediate whose synthesis is controlled by feedback inhibition of Ade4p acting as the purine nucleotide sensor within the cell.In Saccharomyces cerevisiae, biosynthesis pathways are generally negatively regulated by their end product. This regulation usually occurs at two distinct levels, feedback inhibition of an enzyme of the pathway (commonly the first one) and coordinate repression at the transcriptional level of the genes encoding enzymes of the pathway.Studies on the regulation of the purine biosynthesis pathway in S. cerevisiae revealed that all the genes encoding enzymes required for AMP de novo biosynthesis are repressed at the transcriptional level by the presence of extracellular purines (adenine or hypoxanthine) (6,7,10,23). Two transcription factors, named Bas1p and Bas2p, are required for regulated activation of the ADE genes (6) as well as some histidine biosynthesis genes (2,7,35). A LexA-Bas1p fusion can activate a lexAop-lacZ reporter in the presence of Bas2p and in the absence of adenine, suggesting that the regulation process affects the interaction between the two transcription factors (44). A Bas1p subdomain, named BIRD, was identified as being critical for adenine response and Bas1p-Bas2p interaction in vivo (29). However, our understanding of how this domain senses and responds to extracellular adenine is still incomplete.Our previous work on mutants in which purine biosynthesis genes are no longer repressed by extracellular adenine allowed us to better understand the molecular nature of the signal (13). These mutations, named bra for bypass of repression by adenine, define more than nine complementation groups, several of which have been characterized. BRA7 is FCY2, the gene coding for the purine cytosine permease (Fig. 1) (13)....

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. 5Ј-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.

The relationship between amyloid and toxic species is a central problem since the discovery of amyloid structures in different diseases. Despite intensive efforts in the field, the deleterious species remains unknown at the molecular level. This may reflect the lack of any structure-toxicity study based on a genetic approach. Here we show that a structure-toxicity study without any biochemical prerequisite can be successfully achieved in yeast. A PCR mutagenesis of the amyloid domain of HET-s leads to the identification of a mutant that might impair cellular viability. Cellular and biochemical analyses demonstrate that this toxic mutant forms GFP-amyloid aggregates that differ from the wild-type aggregates in their shape, size and molecular organization. The chaperone Hsp104 that helps to disassemble protein aggregates is strictly required for the cellular toxicity. Our structure-toxicity study suggests that the smallest aggregates are the most toxic, and opens a new way to analyze the relationship between structure and toxicity of amyloid species.