Yeast AMP Pathway Genes Respond to Adenine through Regulated Synthesis of a Metabolic Intermediate

Abstract

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)....

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

“…A genetic analysis of mutants unresponsive to adenine has shown that adenine needs to be taken up and metabolized to ADP to exert its regulatory role (Guetsova et al 1997;Rébora et al 2001). This is consistent with the observation that extracellular hypoxanthine downregulates expression of an ADE1-lacZ fusion while guanine has little effect (Guetsova et al 1997) because hypoxanthine can be converted to adenylic nucleotides while guanine cannot ( Figure 15).…”

“…Strikingly, most of the deregulated mutants affected the ADE13 gene encoding adenylosuccinate lyase, suggesting a key role for this step of the pathway ( Figure 15) (Rébora et al 2001). Further genetic analyses revealed that AICAR (59-phosphoribosyl-5-amino-4-imidazole carboxamide) and SAICAR (succinyl-AICAR), the metabolic intermediates just downstream and upstream of Ade13, respectively, play a pivotal role by promoting interactions between Bas1 and Pho2 (Rébora et al 2001(Rébora et al , 2005. Several lines of evidence supported this idea.…”

Regulatory architecture determines optimal regulation of gene expression in metabolic pathways

“…For the third IMA pathway, adenine biosynthesis, it is clear that one (or both) of the two metabolites AICAR and SAICAR induces transcriptional activation via the transcription factor Bas1; however, it is not fully understood which is the major regulator (2). We find that the enzyme Ade17 has a higher induction than any other enzyme in the pathway (Fig.…”

“…To achieve this regulation, a variety of strategies can be used, involving different interplay between metabolites, enzymes, and regulatory proteins. In many cases involving model organisms, the regulatory framework that controls this process is known, and research over past decades has revealed a number of different regulatory strategies (1)(2)(3)(4).…”

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

“…Indeed, apart from playing a crucial role in the control of phosphate homeostasis, Pho2 together with the transcription factor Bas1 co-regulates the expression of the so-called ADE regulon that includes genes involved in the de novo synthesis of purines (Daignan-Fornier and Fink 1992). Consistently, a recent transcriptome analysis revealed that the metabolic intermediate AICAR (5 0 -phosphoribosyl-5-amino-4-imidazole carboxamide), which was previously identified as a transcriptional regulator of the purine pathway genes (Rebora et al 2001;Rebora et al 2005), also mediates the expression of several PHO genes ). These authors reported that Pho4 and Bas1 compete for Pho2 binding and that AICAR stimulates the interaction between either Pho4 or Bas1 with Pho2.…”