Respiring Saccharomyces cerevisiae cells respond to a sudden increase in glucose concentration by a pronounced drop of their adenine nucleotide content. Transient accumulation of the purine salvage pathway intermediate inosine accounts for the apparent loss of adenine nucleotides.Inosine formation in response to perturbations of cellular energy balance depends on the presence of a fermentable carbon source. Under respiratory conditions, AMP accumulates instead and no inosine is formed.Conversion of AXPs into inosine is facilitated by AMP deaminase, Amd1, and IMP-specific 5'-nucleotidase, Isn1. Inosine recycling into the AXP pool is facilitated by the purine nucleoside phosphorylase, Pnp1, and joint action of the phosphoribosyltransferases, Hpt1 and Xpt1.Impaired inosine formation results in altered metabolite pool dynamics in response to glucose addition, but does not change glycolytic flux. However, mutants blocked in inosine formation exhibit delayed growth acceleration after glucose addition.
Yeast cells are exposed to strongly fluctuating nutrient concentrations in their natural environment, which requires rapid and efficient adaptation through rearrangements on all levels of their metabolism. The quantitative understanding of these adaptation processes represents the basis for a directed optimization of the microorganism to suit the needs of biochemical production processes that often impose non-uniform or harsh cultivation conditions (Lara et al, 2006), or require a redirection of metabolic fluxes to improve productivity (Bailey, 1991). In this context, controlled perturbation experiments represent a valuable tool, as they allow studying the transition from one defined physiological state to another under well-characterized conditions. Measurements of metabolite pool dynamics and enzymatic activities in response to different perturbations enable the quantitative mathematical analysis of glycolytic dynamics, which is ultimately meant to discriminate the impact of allosteric regulation and changes in the enzymatic make-up of the cell on the overall metabolic response (Rizzi et al, 1997; Teusink et al, 2000; Daran-Lapujade et al, 2007; van den Brink et al, 2008).
A long-standing problem in the context of these studies was the apparent loss of adenine nucleotides, which immediately follows the relief from glucose limitation (Theobald et al, 1997; Kresnowati et al, 2006). In this study, we showed that the transient accumulation of the purine salvage pathway (PSP) metabolites, IMP and inosine, account for the loss of AXP nucleotides. The pathway for inosine formation and recycling was identified, and the interplay between different pathways during the respiro-fermentative transition is schematically summarized in Figure 10. The presence of a quickly metabolizable sugar causes accumulation of the phosphorylated metabolites F1,6P, T6P and G3P, which results in a transient imbalance of ATP-consuming and ATP-regenerating reactions, and provokes a drop in both ATP and intracellular phosphate (Pi) concentrations. The joint action of fast ATP consumption and the Adk1 reaction results in the net-production of AMP. The accumulation of AMP, however, is prevented by Amd1 and Isn1, which readily convert AMP via IMP into inosine. Recycling of inosine into IMP is facilitated by Pnp1 and the concomitant action of Hpt1 and Xpt1, with Hpt1 having the predominant role. We suggest that inosine formation serves to prevent unscheduled AMP accumulation, and to store AXP nucleotides in a metabolically ‘neutral' form until re-equilibration of glycolysis eventually allows recovery of ATP levels. Recycling of inosine via IMP represents an energy-saving way to replenish the AXP pool, as de novo synthesis of IMP starting from PRPP requires 4 ATP molecules, whereas IMP production from inosine and PRPP is an ATP-neutral process (Figure 10).
In contrast to the behavior observed in the presence of fermentable carbon sources, neither IMP nor inosine were formed in response to perturbations of the cellular energy balance under respiratory conditions. Instead, the drop in ATP level caused a concomitant increase in AMP concentration. Therefore, we conclude that the conversion of AXP nucleotides into inosine represents a specific short-term metabolic response to the perturbation of cellular energy homeostasis, which is controlled by the presence of a fermentable carbon source (or a sugar derivative that can undergo rapid phosphorylation). The discrimination between AMP accumulation and AMP-to-inosine conversion seems to be controlled by Amd1 activity. At the present stage of investigation, we cannot conclude whether regulation of Amd1 is brought about by allosteric control or by posttranslational modification. However, given that inorganic phosphate is a potent inhibitor of Amd1 (Merkler et al, 1989) and intracellular phosphate concentration transiently drops when cells become exposed to fermentable carbon sources (our data, and e.g. Hohmann et al, 1996), phosphate availability is likely to have a pivotal role in the regulation of AXP pool size and inosine formation.
The impact of defective AXP cycling on the global metabolic response to glucose addition was tested under conditions in which respiration was inhibited by antimycin A. The amd1 mutant, in which adenine nucleotide cycling was completely blocked, showed the strongest deviations from the wild-type behavior. In this mutant, adenylic energy charge exhibited a strong drop after glucose addition, and recovered much slower than in wild-type cells. Furthermore, deletion of AMD1 resulted in strong accumulation of AMP and pronounced changes in the dynamics of trehalose-6-phosphate, glycerol-3-phosphate, and PRPP after glucose addition (Figure 6).
Despite pronounced changes in metabolite pool dynamics caused by the deletion of AMD1, no alterations in the production of the fermentative end products, ethanol and glycerol, nor in the consumption of glucose were observed (Figure 9B). However, the amd1 and isn1 mutant strains showed delayed growth acceleration after glucose addition (Figure 9C). The observation of unaltered fermentation capacity but concomitant delay in growth acceleration in the amd and isn1 mutants points to the possibility that regulation of glycolysis is not the major target of AMP. Indeed, our study revealed a delayed increase of PRPP concentration in the amd1 strain after glucose addition. The PRPP protein has a central role as a precursor for purine nucleotide de novo synthesis and the synthesis of amino acids (Vavassori et al, 2005). Hence, limitation of this important precursor may negatively affect growth. In addition, AMP accumulation may have an important signaling function acting, for example, through the cAMP/PKA pathway (Thevelein et al, 2005; Zaman et al, 2009) and/or via Snf1 (Celenza and Carlson, 1984). However, experimental evidence to support this potential link is missing. Thus, the assumption of a signaling role of AMP remains speculative, although intriguing, when asking for the actual function of AXP-to-inosine conversion during the respiro-fermentative transition.
On the basis of this study, we put forward the implication of adenine nucleotide cycling through the PSP in energy homeostasis in yeast. Answering the question whether this pathway is also active in humans could potentially contribute to a better understanding of metabolic processes that control the metabolic transition from respiratory to respiro-fermentative energy supply in muscle upon heavy exercise. In particular, it may help to understand conflicting results on the impaired physical performance of individuals carrying an AMP deaminase dysfunction under these conditions (Tarnopolsky et al, 2001; Fischer et al, 2007).
Respiring Saccharomyces cerevisiae cells respond to a sudden increase in glucose concentration by a pronounced drop of their adenine nucleotide content ([ATP]+[ADP]+[AMP]=[AXP]). The unknown fate of ‘lost' AXP nucleotides represented a long-standing problem for the understanding of the yeast's physiological response to changing growth conditions. Transient accumulation of the purine salvage pathway intermediate, inosine, accounted for the apparent loss of adenine nucleotides. Conversion of AXPs into inosine was facilitated by AMP deaminase, Amd1, and IMP-specific 5′-nucleotidase, Isn1. Inosine recycling into the AXP pool was facilitated by purine nucleoside phosphorylase, Pnp1, and joint action of the phosphoribosyltransferases, Hpt1 and Xpt1. Analysis of changes in 24 intracellular metabolite pools during the respiro-fermentative growth transition in wild-type, amd1, isn1, and pnp1 strains revealed that only the amd1 mutant exhibited significant deviations from the wild-type behavior. Moreover, mutants that were blocked in inosine production exhibited delayed growth acceleration after glucose addition. It is proposed that interconversion of adenine nucleotides and inosine facilitates rapid and energy-cost efficient adaptation of the AXP pool size to changing environmental conditions.