Nuclear versus cytosolic activity of the yeast Hog1 MAP kinase in response to osmotic and tunicamycin-induced ER stress

© 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. We examined the physiological significance of the nuclear versus cytosolic localization of the MAPK Hog1p in the ability of yeast cells to cope with osmotic and ER (endoplasmic reticulum) stress. Our results indicate that nuclear import of Hog1p is not critical for osmoadaptation. Plasma membrane-anchored Hog1p is still able to induce increased expression of GPD1 and glycerol accumulation. This is a key osmoregulatory event, although a small production of the osmolyte coupled with the nuclear import of Hog1p is sufficient to provide osmoresistance. On the contrary, the nuclear activity of Hog1p is dispensable for ER stress adaptation.This research was supported by the Comisión Interministerial de Ciencia y Tecnología project (AGL2010-17516) from the Ministry of Economy and Competitiveness of Spain (MINECO). SG-M was supported by a pre-doctoral fellowship within the “JAE” Program (CSIC).Peer Reviewe

The Formation of hybrid complexes between isoenzymes of glyceraldehyde-3-phosphate dehydrogenase regulates its aggregation state, the glycolytic activity and sphingolipid status in Saccharomyces cerevisiae

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been traditionally considered a housekeeping protein involved in energy generation. However, evidence indicates that GAPDHs from different origins are tightly regulated and that this regulation may be on the basis of glycolysis-related and glycolysis-unrelated functions. In Saccharomyces cerevisiae, Tdh3 is the main GAPDH, although two other isoenzymes encoded by TDH1 and TDH2 have been identified. Like other GAPDHs, Tdh3 exists predominantly as a tetramer, although dimeric and monomeric forms have also been isolated. Mechanisms of Tdh3 regulation may thus imply changes in its oligomeric state or be based in its ability to interact with Tdh1 and/or Tdh2 to form hybrid complexes. However, no direct evidence of the existence of these interactions has been provided and the exact function of Tdh1,2 is unknown. Here, we show that Tdh1,2 immunopurified with a GFP-tagged version of Tdh3 and that lack of this interaction stimulates the Tdh3's aggregation. Furthermore, we found that the combined knockout of TDH1 and TDH2 promotes the loss of cell's viability and increases the growing rate, glucose consumption and CO2 production, suggesting a higher glycolytic flux in the mutant cells. Consistent with this, the tdh3 strain, which displays impaired in vitro GAPDH activity, exhibited the opposite phenotypes. Quite remarkably, tdh1 tdh2 mutant cells show increased sensitivity to aureobasidin A, an inhibitor of the inositolphosphoryl ceramide synthase, while cells lacking Tdh3 showed improved tolerance. The results are in agreement with a link between glycolysis and sphingolipid (SLs) metabolism. Engineering Tdh activity could be thus exploited to alter the SLs status with consequences in different aspects of yeast biotechnology

Sphingolipids and inositol phosphates regulate the tau protein phosphorylation status in humanized yeast

Hyperphosphorylation of protein tau is a hallmark of Alzheimer's disease (AD). Changes in energy and lipid metabolism have been correlated with the late onset of this neurological disorder. However, it is uncertain if metabolic dysregulation is a consequence of AD or one of the initiating factors of AD pathophysiology. Also, it is unclear whether variations in lipid metabolism regulate the phosphorylation state of tau. Here, we show that in humanized yeast, tau hyperphosphorylation is stimulated by glucose starvation in coincidence with the downregulation of Pho85, the yeast ortholog of CDK5. Changes in inositol phosphate (IP) signaling, which has a central role in energy metabolism, altered tau phosphorylation. Lack of inositol hexakisphosphate kinases Kcs1 and Vip1 (IP6 and IP7 kinases in mammals) increased tau hyperphosphorylation. Similar effects were found by mutation of IPK2 (inositol polyphosphate multikinase), or PLC1, the yeast phospholipase C gene. These effects may be explained by IP-mediated regulation of Pho85. Indeed, this appeared to be the case for plc1, ipk2, and kcs1. However, the effects of Vip1 on tau phosphorylation were independent of the presence of Pho85, suggesting additional mechanisms. Interestingly, kcs1 and vip1 strains, like pho85, displayed dysregulated sphingolipid (SL) metabolism. Moreover, genetic and pharmacological inhibition of SL biosynthesis stimulated the appearance of hyperphosphorylated forms of tau, while increased flux through the pathway reduced its abundance. Finally, we demonstrated that Sit4, the yeast ortholog of human PP2A protein phosphatase, is a downstream effector of SL signaling in mediating the tau phosphorylation state. Altogether, our results add new knowledge on the molecular effectors involved in tauopathies and identify new targets for pharmacological intervention

Myriocin-induced adaptive laboratory evolution of an industrial strain of Saccharomyces cerevisiae reveals its potential to remodel lipid composition and heat tolerance

The modification of lipid composition allows cells to adjust membrane biophysical properties in response to changes in environmental temperature. Here, we use adaptive laboratory evolution (ALE) in the presence of myriocin, a sphingolipid (SLs) biosynthesis inhibitor, to remodel the lipid profile of an industrial yeast strain (LH) of Saccharomyces cerevisiae. The approach enabled to obtain a heterogeneous population (LHev) of myriocin-tolerant evolved clones characterized by its growth capacity at high temperature. Myriocin exposure also caused tolerance to soraphen A, an inhibitor of the acetyl-CoA carboxylase Acc1, the rate-limiting enzyme in fatty acid de novo production, supporting a change in lipid metabolism during ALE. In line with this, characterization of two randomly selected clones, LH03 and LH09, showed the presence of lipids with increased saturation degree and reduced acyl length. In addition, the clone LH03, which displays the greater improvement in fitness at 40°C, exhibited higher SL content as compared with the parental strain. Analysis of the LH03 and LH09 genomes revealed a loss of chromosomes affecting genes that have a role in fatty acid synthesis and elongation. The link between ploidy level and growth at high temperature was further supported by the analysis of a fully isogenic set of yeast strains with ploidy between 1N and 4N which showed that the loss of genome content provides heat tolerance. Consistent with this, a thermotolerant evolved population (LH40°) generated from the parental LH strain by heat-driven ALE exhibited a reduction in the chromosome copy number. Thus, our results identify myriocin-driven evolution as a powerful approach to investigate the mechanisms of acquired thermotolerance and to generate improved strains

Heterologous expression of type I antifreeze peptide GS-5 in baker's yeast increases freeze tolerance and provides enhanced gas production in frozen dough

The demand for frozen-dough products has increased notably in the baking industry. Nowadays, no appropriate industrial baker's yeast with optimal gassing capacity in frozen dough is, however, available, and it is unlikely that classical breeding programs could provide significant improvements of this trait. Antifreeze proteins, found in diverse organisms, display the ability to inhibit the growth of ice, allowing them to survive at temperatures below 0°C. In this study a recombinant antifreeze peptide GS-5 was expressed from tne polar fish grubby sculpin (Myoxocephalus aenaeus) in laboratory and industrial baker's yeast strains of Saccharomyces cerevisiae. Production of the recombinant protein increased freezing tolerance in both strains tested. Furthermore, expression of the GS-5 encoding gene enhanced notably the gassing rate and total gas production in frozen and frozen sweet doughs. These effects are unlikely to be due to reduced osmotic damage during freezing/ thawing, because recombinant cells showed growth behavior similar to that of the parent under hypermosmotic stress conditions.This research was supported by the Comisión Interministerial de Ciencia y Tecnología project (PACTI, COO1999AX173) from the Ministry of Science and Technology of Spain. J.P. is supported by a F.P.I. fellowship.Peer Reviewe

Cold response in Saccharomyces cerevisiae: New functions for old mechanisms

The response of yeast cells to sudden temperature downshifts has received little attention compared with other stress conditions. Like other organisms, both prokaryotes and eukaryotes, in Saccharomyces cerevisiae a decrease in temperature induces the expression of many genes involved in transcription and translation, some of which display a cold-sensitivity phenotype. However, little is known about the role played by many cold-responsive genes, the sensing and regulatory mechanisms that control this response or the biochemical adaptations at or near 0°C. This review focuses on the physiological significance of cold-shock responses, emphasizing the molecular mechanisms that generate and transmit cold signals. There is now enough experimental evidence to conclude that exposure to low temperature protects yeast cells against freeze injury through the cold-induced accumulation of trehalose, glycerol and heat-shock proteins. Recent results also show that changes in membrane fluidity are the primary signal triggering the cold-shock response. Notably, this signal is transduced and regulated through classical stress pathways and transcriptional factors, the high-osmolarity glycerol mitogen-activated protein kinase pathway and Msn2/4p. Alternative cold-stress generators and transducers will also be presented and discussed.supported by the Comisión Interministerial de Ciencia y Tecnología projects (AGL2000-0524; AGL2001-1203; AGL2004-462). J.A. is supported by an I3P grant from the Spanish Ministry of Education and Science.Peer Reviewe

Utilización de cepas de Torulaspora delbrueckii en la producción de masas dulces

Referencia OEPM: P200102067.-- Fecha de solicitud: 14/09/2001.-- Titulares: Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Valencia.Utilización de cepas de Torulaspora delbrueckii en la producción de masas dulces. La presente invención consiste en la utilización de cepas de Toulaspora delbrueckii en la producción de masas dulces congeladas. Estas cepas presentan un crecimiento rápido y alto rendimiento de biomasa en medios de melaza acoplado a una buena capacidad fermentativa en masas dulces y saladas, tanto frescas como congeladas. Además, la utilización de estas cepas tiene ventajas adicionales, ya que una sola cepa puede ser utilizada en diferentes tipos de formulaciones panarias, incluidas aquéllas que contienen sal.Peer reviewe

Adaptive evolution of baker's yeast in a dough-like environment enhances freeze and salinity tolerance

12 pages, 7 figures, 2 tables.-- Online version published: 17 Jul 2009.-- The definitive version is available at www3.interscience.wiley.comWe used adaptive evolution to improve freeze tolerance of industrial baker's yeast. Our hypothesis was that adaptation to low temperature is accompanied by enhanced resistance of yeast to freezing. Based on this hypothesis, yeast was propagated in a flour-free liquid dough model system, which contained sorbitol and NaCl, by successive batch refreshments maintained constantly at 12°C over at least 200 generations. Relative to the parental population, the maximal growth rate (µmax) under the restrictive conditions, increased gradually over the time course of the experiment. This increase was accompanied by enhanced freeze tolerance. However, these changes were not the consequence of genetic adaptation to low temperature, a fact that was confirmed by prolonged selection of yeast cells in YPD at 12°C. Instead, the experimental populations showed a progressive increase in NaCl tolerance. This phenotype was likely achieved at the expense of others traits, since evolved cells showed a ploidy reduction, a defect in the glucose derepression mechanism and a loss in their ability to utilize gluconeogenic carbon sources. We discuss the genetic flexibility of S. cerevisiae in terms of adaptation to the multiple constraints of the experimental design applied to drive adaptive evolution and the technologically advantageous phenotype of the evolved population.This research was jointly funded by the Spanish Ministry of Science and Technology (CICYT projects, AGL2004-00462 and AGL2007-65498-C02-01) and the EU's Sixth Framework Program (Marie Curie Reintegration Grants). J.A. was the recipient of a post-doctoral contract within the “I3P” Program from CSIC.Peer reviewe

Redox engineering by ectopic expression of glutamate dehydrogenase genes links NADPH availability and NADH oxidation with cold growth in Saccharomyces cerevisiae

[Background] Cold stress reduces microbial growth and metabolism being relevant in industrial processes like wine making and brewing. Knowledge on the cold transcriptional response of Saccharomyces cerevisiae suggests the need of a proper redox balance. Nevertheless, there are no direct evidence of the links between NAD(P) levels and cold growth and how engineering of enzymatic reactions requiring NAD(P) may be used to modify the performance of industrial strains at low temperature. .[Results] Recombinant strains of S. cerevisiae modified for increased NADPH- and NADH-dependent Gdh1 and Gdh2 activity were tested for growth at low temperature. A high-copy number of the GDH2-encoded glutamate dehydrogenase gene stimulated growth at 15°C, while overexpression of GDH1 had detrimental effects, a difference likely caused by cofactor preferences. Indeed, neither the Trp− character of the tested strains, which could affect the synthesis of NAD(P), nor changes in oxidative stress susceptibility by overexpression of GDH1 and GDH2 account for the observed phenotypes. However, increased or reduced NADPH availability by knock-out or overexpression of GRE3, the NADPH-dependent aldose reductase gene, eliminated or exacerbated the cold-growth defect observed in YEpGDH1 cells. We also demonstrated that decreased capacity of glycerol production impairs growth at 15 but not at 30°C and that 15°C-grown baker’s yeast cells display higher fermentative capacity than those cultivated at 30°C. Thus, increasing NADH oxidation by overexpression of GDH2 would help to avoid perturbations in the redox metabolism induced by a higher fermentative/oxidative balance at low temperature. Finally, it is shown that overexpression of GDH2 increases notably the cold growth in the wine yeast strain QA23 in both standard growth medium and synthetic grape must.[Conclusions] Redox constraints limit the growth of S. cerevisiae at temperatures below the optimal. An adequate supply of NAD(P) precursors as well as a proper level of reducing equivalents in the form of NADPH are required for cold growth. However, a major limitation is the increased need of oxidation of NADH to NAD+ at low temperature. In this scenario, our results identify the ammonium assimilation pathway as a target for the genetic improvement of cold growth in industrial strainsThis research was funded by the Comisión Interministerial de Ciencia y Tecnología (projectAGL2010-17516) from the Ministry of Economy and Competitiveness of Spain (MINECO). LBT was supported by a F. P. I. fellowship. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer Reviewe

Validation of a Flour-Free Model Dough System for Throughput Studies of Baker's Yeast

Evaluation of gene expression in baker's yeast requires the extraction and collection of pure samples of RNA. However, in bread dough this task is difficult due to the complex composition of the system. We found that a liquid model system can be used to analyze the transcriptional response of industrial strains in dough with a high sugar content. The production levels of CO2 and glycerol by two commercial strains in liquid and flour-based doughs were correlated. We extracted total RNA from both a liquid and a flour-based dough. We used Northern blotting to analyze mRNA levels of three stress marker genes, HSP26, GPD1, and ENA1, and 10 genes in different metabolic subcategories. All 13 genes had the same transcriptional profile in both systems. Hence, the model appears to effectively mimic the environment encountered by baker's yeast in high-sugar dough. The liquid dough can be used to help understand the connections between technological traits and biological functions and to facilitate studies of gene expression under commercially important, but experimentally intractable, conditions.Comisión Interministerial de Ciencia y Tecnología project (AGL2001-1203