Resumen de A shared regulatory network allows functional coupling of pho89 and ena1 in response to environmental alkalinization
Microorganisms are exposed to slight changes in the environment, which may imply a stressful situation. The budding yeast Saccharomyces cerevisiae prefers acidic conditions to proliferate; hence, an alkalinization of the external medium impairs its growth. To cope with this stress, S. cerevisiae activates several signaling pathways that create a complex regulatory network, leading to a profound remodeling of the transcriptional profile. The calcineurin pathway, activated by an increase of cytosolic calcium occurring in alkaline conditions, governs gene expression through the transcription factor Crz1. Moreover, high pH stress is sensed by the transmembrane protein Rim21, a component of the Rim101 pathway, which starts a signaling cascade resulting in the proteolytic activation of Rim101. This transcription factor represses the expression of NRG1, another transcriptional repressor, thus indirectly inducing the expression of certain genes. The Snf1 kinase is also activated by alkaline stress and has several targets through which it controls different processes. Among them, Snf1 inhibits the repressors Mig1/Mig2 and Nrg1/Nrg2, alleviating the repression of a diverse group of genes. Furthermore, an increase of the external pH also triggers the PHO signaling pathway, related to phosphate scarcity, causing the inactivation of the Pho80-Pho85 complex and the subsequent accumulation of Pho4 into the nucleus. This transcription factor, along with Pho2, is responsible for the expression of the PHO regulon, the function of which is to maintain the intracellular phosphate pool. The Na+-ATPase-encoding gene ENA1 is an excellent example of coregulation by different signaling pathways. The alkaline induction of this gene depends on Crz1 and on release from the repression by Nrg1, Mig1, and Mig2, triggered by activation of Rim101 and Snf1. Pho89 is a high-affinity sodium/phosphate cotransporter belonging to the PHO regulon. Apart from being upregulated by Pho4, under alkaline conditions PHO89 expression is also dependent on Crz1, being the only PHO gene to display such regulation.
In this work we focus on the regulatory network driving the alkaline induction of PHO89 and its possible role in the high pH stress response. We show that the transcriptional profiles of PHO89 between alkaline and phosphate starvation conditions differ considerably, with a much weaker and delayed expression on the latter. In contrast, the expression of Pho84, the other high-affinity phosphate transporter, is stronger in low phosphate conditions than under alkaline stress. The calcineurin effect on PHO89 is mediated exclusively through Crz1, and this transcription factor is bound to its promoter shortly after the alkaline induction. The Rim101 pathway also upregulates PHO89 during high pH stress by impinging on NRG1. After the alkaline stress, the Snf1 kinase is phosphorylated and mediates the phosphorylation of the repressors Mig1 and Mig2, which exit the nucleus under these same conditions. However, only Mig2 has a relevant role in repressing PHO89. Furthermore, the reg1 mutation, which leads to a hyperactive Snf1, results in increased Pho89 levels over time, indicating the importance of Snf1 in the alkaline expression of this transporter.
In this study we also show the putative relationship between Pho89 and Ena1 in an alkaline environment. Both genes share several regulatory factors which allow their synchronous expression after high pH stress. Interestingly, when cells rely exclusively on Pho89 for phosphate uptake, the absence of ENA1 confers a strong alkali-sensitive phenotype. In this same situation, cells display a substantial increase in their intracellular sodium concentration, suggesting that this might be the cause of impaired growth. These results point towards the idea that the coregulation exhibited by Ena1 and Pho89 could allow their functional coupling, and that the Na+ extrusion ability of Ena1 could serve as a detoxifying mechanism necessary to support continuous Pho89 Na+/Pi cotransport under alkaline conditions.
