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

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

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

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

Faculté de droit de Paris. Thèse pour la licence... par Louis-Jean-François Teste du Baillet, ... [Jus romanum : Deposité vel contra. -Droit français : Du Dépôt.]

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

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A new function for the yeast trehalose-6P synthase (Tps1) protein, as key pro-survival factor during growth, chronological ageing, and apoptotic stress

Looking back to our recent work that challenged the paradigm of trehalose in stress resistance in yeast, our objective was to revisit the role of this disaccharide in chronological life span (CLS), and in the control of apoptosis. Using a catalytically dead variant of the trehalose-6-phosphate synthase (Tps1) protein, (the first enzyme in the trehalose biosynthetic pathway), and by manipulating intracellular trehalose independently of this pathway, we demonstrated that trehalose has no role in CLS or in the inhibition of acetic acid or H 20 2-triggered cell death. We showed instead that, in the absence of any apoptotic stimulus, the Tps1 protein itself was necessary in preventing massive, spontaneous commitment of yeast cells to apoptosis during growth. Without Tps1p, the life span was shortened and cells were sensitized to acetic acid (AA) and H 20 2, whereas the overexpression of the inactive variant of Tps1p almost abolished AA-triggered apoptosis. Genetic interaction analysis of TPS1 and genes such as YCA1, NUC1 and AIF1 indicated that these key executioners of cell death partially relayed tps1Δ-triggered signaling. Our results suggested that the pro-survival role of Tps1p could be connected with its ability to preserve ATP levels in yeast cells

Retraction notice to “A new function for the yeast Trehalose-6P Synthase (Tps1) Protein, as key prosurvivalfactor during growth, chronological ageing, and apoptotic stress”

Withdrawal announcementThis article has been retracted at the request of Marie-Ange Teste, Isabelle Léger-Silvestre, Jean M François andJean-Luc Parrou. Marjorie Petitjean could not be reached.The corresponding author identified major issues, brought them to the attention of the Journal.These issues span from significant errors in the Material and Methods section of the article and major flaws incytometry data analysis to data fabrication on the part of one of the authors.Given these errors, the retracting authors state that the only responsible course of action would be to retract thearticle, to respect scientific integrity and maintain the standards and rigor of literature from the retracting authors'group as well as the Journal.The retracting authors sincerely apologize to the readers and editors

The dual role of amyloid-β-sheet sequences in the cell surface properties of FLO11-encoded flocculins in Saccharomyces cerevisiae

International audienceFungal adhesins (Als) or flocculins are family of cell surface proteins that mediate adhesion to diverse biotic and abiotic surfaces. A striking characteristic of Als proteins originally identified in the pathogenic Candida albicans is to form functional amyloids that mediate cis-interaction leading to the formation of adhesin nanodomains and trans-interaction between amyloid sequences of opposing cells. In this report, we show that flocculins encoded by FLO11 in Saccharomyces cerevisiae behave like adhesins in C. albicans. To do so, we show that the formation of nanodomains under an external physical force requires a threshold number of amyloid-forming sequences in the Flo11 protein. Then, using a genome editing approach, we constructed strains expressing variants of the Flo11 protein under the endogenous FLO11 promoter, leading to the demonstration that the loss of amyloid-forming sequences strongly reduces cell-cell interaction but has no effect on either plastic adherence or invasive growth in agar, both phenotypes being dependent on the N-and C-terminal ends of Flo11p. Finally, we show that the location of Flo11 is not altered either by the absence of amyloid-forming sequences or by the removal of the Nor C-terminus of the protein