Many organisms have evolved survival strategies centred on glucose as their chief cellular carbon and energy source. Cellular glucose availability governs most biological processes such as growth, division, metabolism and the ability to deal with environmental stresses. Our understanding of glucose signalling in eukaryotes has been greatly advanced by studying the model organism Saccharomyces cerevisiae
. Despite its relative simplicity, yeast has developed a complex system to monitor external glucose levels and faithfully relay this information to adjust metabolic and gene expression programmes accordingly. There are in fact several distinct upstream regulatory pathways for glucose regulation, including the Ras/PKA, Gpr1/PKA, Sch9, Yak1, Snf1 and Snf3/Rgt2 signalling pathways, as well as the metabolic pathways (Figure ; for comprehensive reviews, see [1
]). Although transmission of the glucose signal is thought to be redundant [1
], each pathway possesses distinct glucose detection and signal transmission methods.
Figure 1 An overview of the yeast glucose signalling and metabolic pathways. Signalling events are indicated in solid black, metabolic reactions in solid grey lines. Dashed black lines imply glucose signals activating the signalling components, dashed grey lines (more ...)
The Protein Kinase A (PKA) pathway, which is regulated upstream by Ras and Gpr1, is pivotal for the glucose response. In periods of high glucose abundance, it directs the cell to ferment the available glucose to support growth and proliferation, whilst simultaneously repressing the stress response and the use of alternative carbon sources. Ras1 and Ras2 are small monomeric GTPases. In response to high glucose levels, Ras1 and Ras2 are activated and bind to adenylate cyclase, which is composed of Cyr1 and an associated protein Srv2 [4
]. The subsequent increase in cyclic AMP (cAMP) production activates PKA. A GPCR system operates in parallel to Ras [5
]. Upon sensing high glucose levels, Gpr1, the 7-transmembrane receptor, accelerates the GDP for GTP exchange on the Gα-subunit Gpa2, which then activates Cyr1 and thus raises PKA activity. The Sch9 pathway operates in parallel to PKA to couple glucose availability and growth by regulating ribosomal biogenesis and ribosomal protein transcript levels [3
]. The Yak1 and Snf1 signalling pathways are triggered upon depletion of external glucose levels. The protein kinase Yak1 phosphorylates Pop2, part of the Ccr4-Not complex, to regulate transcript levels of stress response and carbohydrate metabolism genes [10
] in a manner antagonistic to PKA [11
]. The kinase Snf1 orchestrates the adaption yeast undergoes upon glucose depletion by mediating derepression of glucose-repressed genes and contributes to the response to other environmental stresses [12
]. Last, the Snf3/Rgt2 signalling pathway consists of the extracellular glucose sensors Snf3 and Rgt2 that modulate the expression of numerous sugar transporter genes (the Hxts, Gal2, Stl1 and Agt1) [14
While the yeast glucose regulatory system has been intensely investigated for decades, with many components and their relationships well defined, numerous aspects remain elusive. Examples include the precise characterisation of connections between the different pathways, determination of the hierarchical organisation of these pathways, as well as establishing the exact contribution of individual components to the overall glucose regulatory system.
Most components of the glucose regulatory system have been assigned to pathways based on a measurable phenotype caused by perturbation of that particular pathway. A classic example is the genetic screen using the sucrose non-fermenting phenotype of yeast mutants, which revealed various components, such as the Snf1 kinase, to be involved in glucose repression [16
]. However, such phenotypes are often specific for individual pathways and hinder systematic comparison of a large number of components from different pathways side by side. Changes at the transcript level underlie many phenotypes. If measured collectively, for example by DNA microarray analysis of deletion mutants, such gene expression profiles can be exploited as detailed molecular phenotypes to systematically characterise many different pathways simultaneously using a single assay [17
]. Similar approaches have previously been applied to analyse the yeast glucose regulatory system [3
], but these studies have been limited to analyses of only a few components. In addition, the use of different strain backgrounds and experimental conditions hinders a systematic comparison between datasets. Here, DNA microarray gene expression profiles of deletion mutants are generated under a standardised high glucose growth condition to obtain a comprehensive overview of the yeast glucose regulatory system. In addition to relating gene expression profiles of pathway members by their similarity, the data is used to link cause and effect by relating the deleted gene to all transcripts significantly changing in response to the deletion [19
]. To fully exploit the data, a new approach is devised that combines both these strategies to infer the underlying transcriptional regulatory network.
Here, we show that the pathways involved in glucose signalling are so tightly interlinked that in effect only one main transcriptional response can be discerned upon disruption of any individual pathway. This response varies in direction to mimic either a high or a low glucose response and reveals both known and unknown relationships within and between individual pathways and their members. In addition, a new network approach uncovers regulatory processes underlying the observed gene expression profiles. The results indicate that pathway members involved in the biosynthesis of the storage carbohydrate trehalose, Tps2 and Tsl1, are the most downstream transcriptional components. The study provides evidence that in response to a perceived alteration in external glucose levels the availability of the storage carbohydrates glycogen and trehalose is regulated, indicative of a shift in the metabolic programme.