Similarities and differences in the biochemical and enzymological properties of the four isomaltases from Saccharomyces cerevisiae

AbstractThe yeast Saccharomyces cerevisiae IMA multigene family encodes four isomaltases sharing high sequence identity from 65% to 99%. Here, we explore their functional diversity, with exhaustive in-vitro characterization of their enzymological and biochemical properties. The four isoenzymes exhibited a preference for the α-(1,6) disaccharides isomaltose and palatinose, with Michaëlis–Menten kinetics and inhibition at high substrates concentration. They were also able to hydrolyze trisaccharides bearing an α-(1,6) linkage, but also α-(1,2), α-(1,3) and α-(1,5) disaccharides including sucrose, highlighting their substrate ambiguity. While Ima1p and Ima2p presented almost identical characteristics, our results nevertheless showed many singularities within this protein family. In particular, Ima3p presented lower activities and thermostability than Ima2p despite only three different amino acids between the sequences of these two isoforms. The Ima3p_R279Q variant recovered activity levels of Ima2p, while the Leu-to-Pro substitution at position 240 significantly increased the stability of Ima3p and supported the role of prolines in thermostability. The most distant protein, Ima5p, presented the lowest optimal temperature and was also extremely sensitive to temperature. Isomaltose hydrolysis by Ima5p challenged previous conclusions about the requirement of specific amino acids for determining the specificity for α-(1,6) substrates. We finally found a mixed inhibition by maltose for Ima5p while, contrary to a previous work, Ima1p inhibition by maltose was competitive at very low isomaltose concentrations and uncompetitive as the substrate concentration increased. Altogether, this work illustrates that a gene family encoding proteins with strong sequence similarities can lead to enzyme with notable differences in biochemical and enzymological properties

MRGI-1, a dominant allele that confers methomyl resistance in yeast expressing the cytoplasmic male sterility T-urfI3 gene from maize

International audienceWe have previously described a eukaryotic het-erologous expression system, with the urf13TW gene in yeast, which mimics the disease susceptibility associated with the Texas cytoplasmic male sterility in maize. This yeast model was used to isolate yeast nuclear mutants conferring methomyl resistance. The genetic strategy we have developed focused on screening for nuclear dominant yeast mutations which restore methomyl resistance. MRGI-1, a yeast nuclear dominant allele, was identified as a methomyl-resistance restorer. We have shown that methomyl resistance co-segregated with a pleiotropic phenotype in the heterozygous MRGI-1/MRG1 diploids, detectable even in the absence of the maize-derived mito-chondrial protein and/or methomyl. We observed an increase in oxygen uptake, a significant decrease of the levels of cytochrome aa 3, and a decrease in the growth yield. This phenotype is influenced by the carbon source and the results suggest a defect in the adaptation to the respiratory pathway in MRGI-1 yeast cells

Effects of various types of stress on the metabolism of reserve carbohydrates in Saccharomyces cerevisiae: genetic evidence for a stress-induced recycling of glycogen and trehalose

International audienceIt is well known that glycogen and trehalose accumulate in yeast under nutrient starvation or entering into the stationary phase of growth, and that high levels of trehalose are found in heat-shocked cells. However, effects of various types of stress on trehalose, and especially on glycogen, are poorly documented. Taking into account that almost all genes encoding the enzymes involved in the metabolism of these two reserve carbohydrates contain between one and several copies of the stress-responsive element (STRE), an investigation was made of the possibility of a link between the potential transcriptional induction of these genes and the accumulation of glycogen and trehalose under different stress conditions. Using transcriptional fusions, it was found that all these genes were induced in a similar fashion, although to various extents, by temperature, osmotic and oxidative stresses. Experiments performed with an msn2/msn4 double mutant proved that the transcriptional induction of the genes encoding glycogen synthase (GSY2) and trehalose-6- phosphate synthase (TPS1) was needed for the small increase in glycogen and trehalose upon exposure to a mild heat stress and salt shock. However, the extent of transcriptional activation of these genes upon stresses in wild-type strains was not correlated with a proportional rise in either glycogen or trehalose. The major explanation for this lack of correlation comes from the fact that genes encoding the enzymes of the biosynthetic and of the biodegradative pathways were almost equally induced. Hence, trehalose and glycogen accumulated to much higher levels in cells lacking neutral trehalase or glycogen phosphorylase exposed to stress conditions, which suggested that one of the major effects of stress in yeast is to induce a wasteful expenditure of energy by increasing the recycling of these molecules. We also found that transcriptional induction of STRE-controlled genes was abolished at temperatures above 40 °C, while induction was still observed for a heat-shockelement- regulated gene. Remarkably, trehalose accumulated to very high levels under this condition. This can be explained by a stimulation of trehalose synthase and inhibition of trehalase by high temperature

Yeast tolerance to various stresses relies on the trehalose-6P synthase (Tps1) protein, not on trehalose: WITHDRAWAL notice in J Biol Chem 2019 Apr 12;294(15):5812. doi: 10.1074/jbc.W119.008564.

Background: Decades of observations strengthened the idea that trehalose is a chemical chaperone. Results: A catalytically inactive variant of the trehalose-6P synthase (Tps1) maintains cell survival and energy homeostasis under stress exposure. Conclusion: The Tps1 protein itself, not trehalose, is crucial for cell integrity. Significance: This work provides unbiased evidence for an alternative function of Tps1, a new moonlighting protein. Trehalose is a stable disaccharide commonly found in nature, from bacteria to fungi and plants. For the model yeast Saccharomyces cerevisiae, claims that trehalose is a stress protectant were based indirectly either on correlation between accumulation of trehalose and high resistance to various stresses or on stress hypersensitivity of mutants deleted for TPS1, which encodes the first enzyme in trehalose biosynthetic pathway. Our goal was to investigate more directly which one, between trehalose and/or the Tps1 protein, may serve yeast cells to withstand exposure to stress. By employing an original strategy that combined the use of mutant strains expressing catalytically inactive variants of Tps1, with MAL(+) yeast strains able to accumulate trehalose from an exogenous supply, we bring for the first time unbiased proof that trehalose does not protect yeast cells from dying and that the stress-protecting role of trehalose in this eukaryotic model was largely overestimated. Conversely, we identified the Tps1 protein as a key player for yeast survival in response to temperature, oxidative, and desiccation stress. We also showed by robust RT-quantitative PCR and genetic interaction analysis that the role of Tps1 in thermotolerance is not dependent upon Hsf1-dependent transcription activity. Finally, our results revealed that the Tps1 protein is essential to maintain ATP levels during heat shock. Altogether, these findings supported the idea that Tps1 is endowed with a regulatory function in energy homeostasis, which is essential to withstand adverse conditions and maintain cellular integrity

The Saccharomyces cerevisiae YPR184w gene encodes the glycogen debranching enzyme.

International audienceThe 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

Yeast tolerance to various stresses relies on the Trehalose-6P Synthase (Tps1) protein, not on trehalose

Yeast tolerance to various stresses relies on the Trehalose-6P Synthase (Tps1) protein, not on trehalose. 27. International Conference on Yeast Genetics and Molecular Biology (ICYGMB

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

International audienceGenes 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 transcrip-tional 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

Genomic and structural comparative analysis of trehalose-6-phosphate synthase protein

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Validation of reference genes for quantitative expression analysis by real-time RT-PCR in <it>Saccharomyces cerevisiae</it>

<p>Abstract</p> <p>Background</p> <p>Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as <it>ACT1</it>, <it>RDN18 </it>or <it>PDA1 </it>are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in <it>Saccharomyces cerevisiae</it>.</p> <p>Results</p> <p>From public microarray datasets, we selected potential reference genes whose expression remained apparently invariable during long-term growth on glucose. Using the algorithm geNorm, <it>ALG9</it>, <it>TAF10</it>, <it>TFC1 </it>and <it>UBC6 </it>turned out to be genes whose expression remained stable, independent of the growth conditions and the strain backgrounds tested in this study. We then showed that the geometric averaging of any subset of three genes among the six most stable genes resulted in very similar normalized data, which contrasted with inconsistent results among various biological samples when the normalization was performed with <it>ACT1</it>. Normalization with multiple selected genes was therefore applied to transcriptional analysis of genes involved in glycogen metabolism. We determined an induction ratio of 100-fold for <it>GPH1 </it>and 20-fold for <it>GSY2 </it>between the exponential phase and the diauxic shift on glucose. There was no induction of these two genes at this transition phase on galactose, although in both cases, the kinetics of glycogen accumulation was similar. In contrast, <it>SGA1 </it>expression was independent of the carbon source and increased by 3-fold in stationary phase.</p> <p>Conclusion</p> <p>In this work, we provided a set of genes that are suitable reference genes for quantitative gene expression analysis by real-time RT-PCR in yeast biological samples covering a large panel of physiological states. In contrast, we invalidated and discourage the use of <it>ACT1 </it>as well as other commonly used reference genes (<it>PDA1, TDH3, RDN18</it>, etc) as internal controls for quantitative gene expression analysis in yeast.</p

Withdrawal: Yeast tolerance to various stresses relies on the trehalose-6P synthase (Tps1) protein, not on trehalose.

International audienceThis article has been withdrawn by Marie-Ange Teste, Jean M. François, and Jean-Luc Parrou. Marjorie Petitjean could not be reached. The corresponding author identified major issues and brought them to the attention of the Journal. These issues span significant errors in the Materials and Methods section of the article and major flaws in cytometry data analysis to data fabrication on the part of one of the authors. Given these errors, the withdrawing authors state that the only responsible course of action would be to withdraw the article to respect scientific integrity and maintain the standards and rigor of literature from the withdrawing authors' group as well as the Journal. The withdrawing authors sincerely apologize to the readers and editors