Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae.

Glycogen and trehalose are the two glucose stores of yeast cells. The large variations in the cell content of these two compounds in response to different environmental changes indicate that their metabolism is controlled by complex regulatory systems. In this review we present information on the regulation of the activity of the enzymes implicated in the pathways of synthesis and degradation of glycogen and trehalose as well as on the transcriptional control of the genes encoding them. cAMP and the protein kinases Snf1 and Pho85 appear as major actors in this regulation. From a metabolic point of view, glucose-6-phosphate seems the major effector in the net synthesis of glycogen and trehalose. We discuss also the implication of the recently elucidated TOR-dependent nutrient signalling pathway in the control of the yeast glucose stores and its integration in growth and cell division. The unexpected roles of glycogen and trehalose found in the control of glycolytic flux, stress responses and energy stores for the budding process, demonstrate that their presence confers survival and reproductive advantages to the cell. The findings discussed provide for the first time a teleonomic value for the presence of two different glucose stores in the yeast cell.

The Saccharomyces cerevisiae YPR184w gene encodes the glycogen debranching enzyme.

The YPR184w gene encodes a 1536-amino acid protein that is 34-39% identical to the mammal, Drosophila melanogaster and Caenorhabditis elegans glycogen debranching enzyme. The N-terminal part of the protein possesses the four conserved sequences of the alpha-amylase superfamily, while the C-terminal part displays 50% similarity with the C-terminal of other eukaryotic glycogen debranching enzymes. Reliable measurement of alpha-1,4-glucanotransferase and alpha-1, 6-glucosidase activity of the yeast debranching enzyme was determined in strains overexpressing YPR184w. The alpha-1, 4-glucanotransferase activity of a partially purified preparation of debranching enzyme preferentially transferred maltosyl units than maltotriosyl. Deletion of YPR184w prevents glycogen degradation, whereas overexpression had no effect on the rate of glycogen breakdown. In response to stress and growth conditions, the transcriptional control of YPR184w gene, renamed GDB1 (for Glycogen DeBranching gene), is strictly identical to that of other genes involved in glycogen metabolism.

A Ralstonia solanacearum type III effector directs the production of the plant signal metabolite trehalose-6-phosphate.

UNLABELLED: The plant pathogen Ralstonia solanacearum possesses two genes encoding a trehalose-6-phosphate synthase (TPS), an enzyme of the trehalose biosynthetic pathway. One of these genes, named ripTPS, was found to encode a protein with an additional N-terminal domain which directs its translocation into host plant cells through the type 3 secretion system. RipTPS is a conserved effector in the R. solanacearum species complex, and homologues were also detected in other bacterial plant pathogens. Functional analysis of RipTPS demonstrated that this type 3 effector synthesizes trehalose-6-phosphate and identified residues essential for this enzymatic activity. Although trehalose-6-phosphate is a key signal molecule in plants that regulates sugar status and carbon assimilation, the disruption of ripTPS did not alter the virulence of R. solanacearum on plants. However, heterologous expression assays showed that this effector specifically elicits a hypersensitive-like response on tobacco that is independent of its enzymatic activity and is triggered by the C-terminal half of the protein. Recognition of this effector by the plant immune system is suggestive of a role during the infectious process. IMPORTANCE: Ralstonia solanacearum, the causal agent of bacterial wilt disease, infects more than two hundred plant species, including economically important crops. The type III secretion system plays a major role in the pathogenicity of this bacterium, and approximately 70 effector proteins have been shown to be translocated into host plant cells. This study provides the first description of a type III effector endowed with a trehalose-6-phosphate synthase enzymatic activity and illustrates a new mechanism by which the bacteria may manipulate the plant metabolism upon infection. In recent years, trehalose-6-phosphate has emerged as an essential signal molecule in plants, connecting plant metabolism and development. The finding that a bacterial pathogen could induce the production of trehalose-6-phosphate in plant cells further highlights the importance of this metabolite in multiple aspects of the molecular physiology of plants.

STRE- and cAMP-independent transcriptional induction of Saccharomyces cerevisiae GSY2 encoding glycogen synthase during diauxic growth on glucose.

It has been shown that the so-called stationary phase GSY2 gene encoding glycogen synthase was induced as the cells left the exponential phase of growth, while glucose and all other nutrients were still plentiful in the medium (Parrou et al., 1999). Since this effect was essentially controlled at the transcriptional level, we looked for the cis- and trans-acting elements required for this specific growth-related genetic event. We demonstrated that mutations of the HAP2/3/4 binding site and of the two STress-Responsive cis-Elements (STRE) did not abolish the early induction of GSY2, although the latter mutation led to a 20-fold drop in the transcriptional activity of the promoter, as determined from lacZ gene fusions. Insertion of a DNA fragment (from -390 to -167 bp, relative to the ATG) of the promoter lacking the two STREs, upstream to the TATA box of a CYC1-lacZ fusion gene, allowed this reporter gene to be induced with a kinetic similar to that of GSY2-lacZ. Mutations in BCY1, which results in a hyperactive protein kinase A, did not alleviate the early induction, while causing a five- to 10-fold reduction in the transcriptional activity of GSY2. In addition, the repressive effect of protein kinase A was quantitatively conserved when both STREs were mutated in GSY2 promoter, indicating that the negative control of gene expression by the RAS-cAMP signalling pathway does not act solely through STREs. Taken together, these results are indicative of an active process that couples growth control to dynamic glucose consumption.

Combinatorial control by the protein kinases PKA, PHO85 and SNF1 of transcriptional induction of the Saccharomyces cerevisiae GSY2 gene at the diauxic shift.

Genes involved in storage carbohydrate metabolism are coordinately induced when yeast cells are subjected to conditions of stress, or when they exit the exponential growth phase on glucose. We show that the STress Responsive Elements (STREs) present in the promoter of GSY2 are essential for gene activation under conditions of stress, but dispensable for gene induction and glycogen accumulation at the diauxic shift on glucose. Using serial promoter deletion, we found that the latter induction could not be attributed to a single cis -regulatory sequence, and present evidence that this mechanism depends on combinatorial transcriptional control by signalling pathways involving the protein kinases Pho85, Snf1 and PKA. Two contiguous regions upstream of the GSY2 coding region are necessary for negative control by the cyclin-dependent protein kinase Pho85, one of which is a 14-bp G/C-rich sequence. Positive control by Snf1 is mediated by Mig1p, which acts indirectly on the distal part of the GSY2 promoter. The PKA pathway has the most pronounced effect on GSY2, since transcription of this gene is almost completely abolished in an ira1ira2 mutant strain in which PKA is hyperactive. The potent negative effect of PKA is dependent upon a branched pathway involving the transcription factors Msn2/Msn4p and Sok2p. The SOK2 branch was found to be effective only under conditions of high PKA activity, as in a ira1ira2 mutant, and this effect was independent of Msn2/4p. The Msn2/4p branch, on the other hand, positively controls GSY2 expression directly through the STREs, and indirectly via a factor that still remains to be discovered. In summary, this study shows that the transcription of GSY2 is regulated by several different signalling pathways which reflect the numerous factors that influence glycogen synthesis in yeast, and suggests that the PKA pathway must be deactivated to allow gene induction at the diauxic shift.

Dynamic responses of reserve carbohydrate metabolism under carbon and nitrogen limitations in Saccharomyces cerevisiae.

The dynamic responses of reserve carbohydrates with respect to shortage of either carbon or nitrogen source was studied to obtain a sound basis for further investigations devoted to the characterization of mechanisms by which the yeast Saccharomyces cerevisiae can cope with nutrient limitation during growth. This study was carried out in well-controlled bioreactors which allow accurate monitoring of growth and frequent sampling without disturbing the culture. Under glucose limitation, genes involved in glycogen and trehalose biosynthesis (GLG1, GSY1, GSY2, GAC1, GLC3, TPS1), in their degradation (GPH1, NTHI), and the typical stress-responsive CTT1 gene were coordinately induced in parallel with glycogen, when the growth has left the pure exponential phase and while glucose was still plentiful in the medium. Trehalose accumulation was delayed until the diauxic shift, although TPS1 was induced much earlier, due to hydrolysis of trehalose by high trehalase activity. In contrast, under nitrogen limitation, both glycogen and trehalose began to accumulate at the precise time when the nitrogen source was exhausted from the medium, coincidentally with the transcriptional activation of genes involved in their metabolism. While this response to nitrogen starvation was likely mediated by the stress-responsive elements (STREs) in the promoter of these genes, we found that these elements were not responsible for the co-induction of genes involved in reserve carbohydrate metabolism during glucose limitation, since GLG1, which does not contain any STRE, was coordinately induced with GSY2 and TPS1.

AGT1, encoding an alpha-glucoside transporter involved in uptake and intracellular accumulation of trehalose in Saccharomyces cerevisiae.

The trehalose content in Saccharomyces cerevisiae can be significantly manipulated by including trehalose at an appropriate level in the growth medium. Its uptake is largely dependent on the expression of AGT1, which encodes an alpha-glucoside transporter. The trehalose found in a tps1 mutant of trehalose synthase may therefore largely reflect its uptake from the enriched medium that was employed.

Pig microsatellites isolated from cosmids revealing polymorphism and localized on chromosomes.

One of the most widely studied simple sequences in the mammalian genome is the (TG)n dinucleotide sequence. Because these microsatellites are highly polymorphic, we chose to study microsatellites from cosmids to provide genetic markers for the porcine genome. After screening a porcine cosmid library with a (CA)10 probe, 20 cosmids containing microsatellites were subcloned and 17 microsatellites identified by sequencing. Oligonucleotide primers flanking the repeat were designed for seven (TG)n microsatellites with n > 14. These seven microsatellites revealed polymorphism and were regionally assigned to chromosomes by fluorescent in situ hybridization of initial cosmids. These seven loci will be useful for both the construction of the genetic map and as landmark loci on the physical map of the porcine genome.