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Macroautophagy/autophagy is an essential adaptive physiological response in eukaryotes induced during nutrient starvation, including glucose, the primary immediate carbon and energy source for most cells. Although the molecular mechanisms that induce autophagy during glucose starvation have been extensively explored in the budding yeast

The survival of eukaryotic organisms during environmental changes is largely dependent on the adaptive responses elicited by signal transduction cascades, including those regulated by the Mitogen-Activated Protein Kinase (MAPK) pathways. The Cell Integrity Pathway (CIP), one of the three MAPK pathways found in the simple eukaryote fission of yeast Schizosaccharomyces pombe, shows strong homology with mammalian Extracellular signal-Regulated Kinases (ERKs). Remarkably, studies over the last few decades have gradually positioned the CIP as a multi-faceted pathway that impacts multiple functional aspects of the fission yeast life cycle during unperturbed growth and in response to stress. They include the control of mRNA-stability through RNA binding proteins, regulation of calcium homeostasis, and modulation of cell wall integrity and cytokinesis. Moreover, distinct evidence has disclosed the existence of sophisticated interplay between the CIP and other environmentally regulated pathways, including Stress-Activated MAP Kinase signaling (SAPK) and the Target of Rapamycin (TOR). In this review we present a current overview of the organization and underlying regulatory mechanisms of the CIP in S. pombe, describe its most prominent functions, and discuss possible targets of and roles for this pathway. The evolutionary conservation of CIP signaling in the dimorphic fission yeast S. japonicus will also be addressed.

Quorum sensing (QS) has been described in prokaryotes as a mechanism that generates coordinated responses dependent on cell population density. Autoinducers, also named quorum-sensing molecules (QSMs), are accumulated into the medium in parallel to the increase of cell population density. Later work has also established its presence in a number of pathogenic and nonpathogenic filamentous fungi as well as in yeast cells. QSMs have a crucial impact in major aspects of fungal development, including morphogenesis, sexual differentiation, consortia behavior, and apoptosis. Significantly, QS is closely linked to fungal pathogenesis by modulating yeast to hyphae transition and biofilm assembly. In this chapter, we describe current advances in QS mechanisms in fungal species and also discuss the potential use of QSMs in biotechnological applications and as antifungal agents.

The final step in post-translational processing of Ras and Rho GTPases involves methylation of the prenylated cysteine residue by an isoprenylcysteine-O-carboxyl methyltransferase (ICMT). ICMT activity is essential for cell growth and development in higher eukaryotes, and inhibition of GTPase methylation has become an attractive target in cancer therapy to inactivate prenylated oncoproteins. However, the specificity and dynamics of the GTPase methylation process remain to be fully clarified. Notably, cells lacking Mam4, the ICMT ortholog in the fission yeast Schizosaccharomyces pombe, are viable. We have exploited this feature to analyze the role of methylation on GTPase localization and function. We show that methylation differentially affects GTPase membrane localization, being particularly relevant for plasma membrane tethering and downstream signaling of palmitoylated and farnesylated GTPases Ras1 and Rho2 lacking C-terminal polybasic motifs. Indeed, Ras1 and Rho2 cysteine methylation is required for proper regulation of differentiation elicited by MAPK Spk1 and for stress-dependent activation of the cell integrity pathway (CIP) and its main effector MAPK Pmk1. Further, Mam4 negatively regulates TORC2 signaling by a cross-inhibitory mechanism relying on Rho GTPase methylation. These results highlight the requirement for a tight control of GTPase methylation in vivo to allow adequate GTPase function., This work was supported by grants from Ministerio de Economía y Competitividad (BFU2014-52828-P and BIO2015-69958-P), and Fundación Séneca (19246/PI/14), Spain. European Regional Development Fund (ERDF) co-funding from the European Union.

Cytokinesis, which enables the physical separation of daughter cells once mitosis has been completed, is executed in fungal and animal cells by a contractile actin- and myosin-based ring (CAR). In the fission yeast Schizosaccharomyces pombe, the formin For3 nucleates actin cables and also co-operates for CAR assembly during cytokinesis. Mitogen-activated protein kinases (MAPKs) regulate essential adaptive responses in eukaryotic organisms to environmental changes. We show that the stress-activated protein kinase pathway (SAPK) and its effector, MAPK Sty1, downregulates CAR assembly in S. pombe when its integrity becomes compromised during cytoskeletal damage and stress by reducing For3 levels. Accurate control of For3 levels by the SAPK pathway may thus represent a novel regulatory mechanism of cytokinesis outcome in response to environmental cues. Conversely, SAPK signaling favors CAR assembly and integrity in its close relative Schizosaccharomyces japonicus, revealing a remarkable evolutionary divergence of this response within the fission yeast clade., This work was supported by the Ministerio de Ciencia, Innovación y Universidades, Spain [Grant references BFU2017-82423-P and PGC2018-098924-B-100], Fundación Séneca de la Región de Murcia [Grant reference 20856/PI/18], and Junta de Castilla y León ‘Escalera de la Excelencia’, Spain [Grant reference CLU-2017–03]. European Regional Development Fund (ERDF) co-funding from the European Union. E G-G and F P-R are Formación de Profesorado Universitario (FPU) pre-doctoral fellows from the University of Murcia and the Ministerio de Ciencia, Innovación y Universidades, Spain, respectively.

Quorum sensing (QS), a mechanism of microbial communication dependent on cell density, governs developmental decisions in many bacteria and in some pathogenic and non-pathogenic fungi including yeasts. In these simple eukaryotes this response is mediated by the release into the growth medium of quorum-sensing molecules (QSMs) whose concentration increases proportionally to the population density. To date the occurrence of QS is restricted to a few yeast species. We show that a QS mediated by the aromatic alcohols phenylethanol and tryptophol represses the dimorphic yeast to hypha differentiation in the fission yeast S. japonicus in response to an increased population density. In addition, the stress activated MAPK pathway (SAPK), which controls cell cycle progression and adaptation to environmental changes in this organism, constitutively represses yeast to hypha differentiation both at transcriptional and post-translational levels. Moreover, deletion of its main effectors Sty1 MAPK and Atf1 transcription factor partially suppressed the QS-dependent block of hyphal development under inducing conditions. RNAseq analysis showed that the expression of nrg1+, which encodes a putative ortholog of the transcription factor Nrg1 that represses yeast to hypha dimorphism in C. albicans, is downregulated both by QS and the SAPK pathway. Remarkably, Nrg1 may act in S. japonicus as an activator of hyphal differentiation instead of being a repressor. S. japonicus emerges as an attractive and amenable model organism to explore the QS mechanisms that regulate cellular differentiation in fungi., Author summary Quorum sensing is a relevant mechanism of communication dependent on population density that controls cell development and pathogenesis in microorganisms including fungi. We describe a quorum sensing mediated by the release of aromatic alcohols in the growth medium that blocks hyphal development in the fission yeast Schizosaccharomyces japonicus. This is the first description of such a mechanism in the fission yeast lineage, and confirms its expansion along Ascomycota fungi. The stress-responsive pathway (SAPK), which regulates fungal growth and differentiation, limits hyphal growth in S. japonicus in a constitutive fashion, and nonfunctional SAPK mutants are partially insensitive to quorum sensing and able to form hyphae in high cell density cultures. Nrg1, an important factor that blocks hyphal development in the pathogen Candida albicans, activates hyphal growth in S. japonicus, and its expression is counteracted by both quorum sensing and the SAPK pathway. Nrg1 function may thus have diverged evolutionary in this organism from being a repressor to an activator of hyphal development. S. japonicus emerges as a suitable model organism to explore the intricate mechanisms regulating fungal differentiation.

The two PKC orthologs Pck1 and Pck2 in the fission yeast Schizosaccharomyces pombe operate in a redundant fashion to control essential functions, including morphogenesis and cell wall biosynthesis, as well as the activity of the cell integrity pathway and its core element, the MAPK Pmk1. We show here that, despite the strong structural similarity and functional redundancy of these two enzymes, the mechanisms regulating their maturation, activation, and stabilization have a remarkably distinct biological impact on both kinases. We found that, in contrast to Pck2, putative in vivo phosphorylation of Pck1 within the conserved activation loop, turn, and hydrophobic motifs is essential for Pck1 stability and biological functions. Constitutive Pck activation promoted dephosphorylation and destabilization of Pck2, whereas it enhanced Pck1 levels to interfere with proper downstream signaling to the cell integrity pathway via Pck2. Importantly, although catalytic activity was essential for Pck1 function, Pck2 remained partially functional independent of its catalytic activity. Our findings suggest that early divergence from a common ancestor in fission yeast involved important changes in the mechanisms regulating catalytic activation and stability of PKC family members to allow for flexible and dynamic control of downstream functions, including MAPK signaling., This work was supported by Ministerio de Economía y Competitividad Grants BFU2014-52828-P and BIO2015-69958-P, Fundación Séneca Grant 19246/PI/14, and European Regional Development Fund co-funding from the European Union.

The activation of neutral trehalase (Ntp1) by metabolic and physical stresses in Schizosaccharomyces pombe is dependent on protein kinases Pka1 or Sck1. Mutant ntp1 alleles altered for potentially phosphorylatable serine residues within the regulatory domain of the enzyme were integrated under the control of the native promoter in an ntp1-deleted background. The trehalase variants were expressed to a level similar to that of wild type trehalase from control cells. Wild type trehalase protein accumulated and became activated upon stress while a single change in the evolutionary conserved perfect consensus site for Pka1-dependent phosphorylation (Ser71), as well as point mutations in two other putative phosphorylation sites (Ser6, Ser51), produced inactive trehalases unresponsive to stress. Trehalose content in the trehalase mutated strains increased upon salt stress to a level comparable to that shown by an ntp1-deleted mutant. When exposed to heat shock, trehalose hyperaccumulated in the ntp1-null strain lacking trehalase protein and this phenotype was shown by some (Ser71), but not all, strains with serine mutated trehalases. The mutant trehalases retained the ability to form complexes with trehalose-6-phosphate synthase. These data support a role of potentially phosphorylated specific sites for the activation of S. pombe neutral trehalase and for the heat shock-induced accumulation of trehalose.

In the fission yeast Schizosaccharomyces pombe, a heat shock enhances transcription of the ntp1+ gene, encoding the hydrolytic enzyme neutral trehalase. As compared to wild-type cells, cells devoid of the MAP kinase Sty1p showed a strong decrease in ntp1+ expression induced by the temperature upshift, indicating that the stress-activated protein kinase (SAPK) pathway regulates the expression of this gene during heat shock. The transcription factor Atf1p, which is the main downstream target for Sty1p in the SAPK pathway, appears to be involved in such control, since ntp1+ expression under heat shock proved to be significantly blocked in atf1+-disrupted cells. Serial deletion and point mutation analyses of the ntp1+ promoter, as well as electrophoretic mobility shift assays, revealed the existence of a CRE-like element as the target for Atf1p-mediated expression under thermal stress. The relevance of two putative HSE elements located in the ntp1+ promoter was also investigated for their potential role in regulating ntp1+ transcription during heat shock. The results support a model in which heat-induced Atf1p binding to the CRE-like element favours the subsequent interaction of the heat shock factor (HSF) with HSE elements in the ntp1+ promoter. Unlike what happens under osmostress or oxidative treatments, Sty1p has no role in the post-translational activation of neutral trehalase induced by heat shock in the fission yeast. Copyright © 2004 John Wiley & Sons, Ltd.

Neutral trehalases mobilize trehalose accumulated by fungal cells as a protective and storage carbohydrate. A structural feature of these enzymes is the presence of an EF-like motif similar to that shown by many Ca2+-binding proteins. In this study we provide direct evidence for physical binding of Ca2+ to neutral trehalase (Ntp1p) of the fission yeast Schizosaccharomyces pombe, and show that aspartic residues at positions 97 and 108 in the conserved putative Ca2+-binding motif of Ntp1p appear to be responsible for this interaction. Mutations in these residues do not interfere with the ability of Ntp1p to associate in vivo with trehalose-6-phosphate synthase, but prevent activation of neutral trehalase triggered by the addition of glucose or by subjecting cells to stressing conditions. Strains expressing Ntp1p variants that are unable to bind Ca2+ partially resemble those devoid of the ntp1+ gene in terms of trehalose hyperaccumulation. Gel filtration of cell extracts from wild-type cells after EDTA treatment or from cells containing Ntp1p with mutations in aspartic acid residues within the Ca2+-binding site revealed that Ntp1p eluted mainly in an inactive conformation instead of the dimeric or trimeric active form of the enzyme. These results suggest that activation of S. pombe Ntp1p under different conditions depends upon Ca2+ binding through the Ca2+-binding motif as a prerequisite for correct enzyme oligomerization to its active form. Given the high degree of conservation of the Ca2+ accommodation site, this might be a general mechanism regulating neutral trehalase activity in other yeasts and filamentous fungi.

In the fission yeast Schizosaccharomyces pombe the Wak1p/Win1p-Wis1p-Sty1p stress-activated protein kinase (SAPK) pathway relays environmental signals to the transcriptional machinery and modulates gene expression via a cascade of protein phosphorylation. Cells of S. pombe subjected to cold shock (transfer from 28 °C to 15 °C) transiently activated the Sty1p mitogen-activated protein kinase (MAPK) by phosphorylation. Induction of this response was completely abolished in cells disrupted in the upstream response regulator Mcs4p. The cold-triggered Sty1p activation was partially dependent on Wak1p MAPKKK and fully dependent on Wis1p MAPKK suggesting that the signal transmission follows a branched pathway, with the redundant MAPKKK Win1p as alternative transducer to Wis1p, which subsequently activates the effector Sty1p MAPK. Also, the bZIP transcription factor Atf1p became phosphorylated in a Sty1p-dependent way during the cold shock and this phosphorylation was found responsible for the increased expression of gpd1+, ctt1+, tps1+ and ntp1+ genes. Strains deleted in transcription factors Atf1p or Pcr1p were unable to grow upon incubation at low temperature whereas those disrupted in any member of the SAPK pathway were able to do so. These data reveal that S. pombe responds to cold by inducing the SAPK pathway. However, such activation is dispensable for yeast growth in cold conditions, supporting that the presence of Atf1/Pcr1 heterodimers, rather than an operative SAPK pathway, is critical to ensure yeast growth at low temperature by an as yet undefined mechanism.

Trehalose metabolism is an essential component of the stress response in yeast cells. In this work we show that the products of the principal genes involved in trehalose metabolism in Schizosaccharomyces pombe, tps1 + (coding for trehalose6-P synthase, Tps1p), ntp1 + (encoding neutral trehalase, Ntp1p) and tpp1 + (that codes for trehalose-6-P phosphatase, Tpp1p), interact in vitro with each other and with themselves to form protein complexes. Disruption of the gene tps1 + blocks the activation of the neutral trehalase induced by heat shock but not by osmotic stress. We propose that this association may reflect the Tps1p-dependent requirement for thermal activation of trehalase. Data reported here indicate that following a heat shock the enzyme activity of trehalase is associated with Ntp1p dimers or trimers but not with either Ntp1p monomers or with complexes involving Tps1p. These results raise the possibility that heat shock and osmotic stress activate trehalase differentially by acting in the first case through an specific mechanism involving Tps1p–Ntp1p complexes. This study provides the first evidence for the participation of the catabolic enzyme trehalase in the structural framework of a regulatory macromolecular complex containing trehalose6-P synthase in the fission yeast.

The fission yeast protein kinase C (PKC) ortholog Pck2 controls cell wall synthesis and is a major upstream activator of the cell integrity pathway (CIP) and its core component, the MAP kinase Pmk1 (also known as Spm1), in response to environmental stimuli. We show that in vivo phosphorylation of Pck2 at the conserved T842 activation loop during growth and in response to different stresses is mediated by the phosphoinositide-dependent kinase (PDK) ortholog Ksg1 and an autophosphorylation mechanism. However, T842 phosphorylation is not essential for Pmk1 activation, and putative phosphorylation at T846 might play an additional role in Pck2 catalytic activation and downstream signaling. These events, together with turn motif autophosphorylation at T984 and binding to small GTPases Rho1 and/or Rho2, stabilize Pck2 and render it competent to exert its biological functions. Remarkably, the target of rapamycin complex 2 (TORC2) does not participate in the catalytic activation of Pck2, but instead contributes to de novo Pck2 synthesis, which is essential to activate the CIP in response to cell wall damage or glucose exhaustion. These results unveil a novel mechanism whereby TOR regulates PKC function at a translational level, and they add a new regulatory layer to MAPK signaling cascades.

We have characterized an open reading frame of 2,454 bp on chromosome I of Schizosaccharomyces pombe as the gene encoding trehalose-6P phosphatase ( tpp1 + ). Disruption of tpp1 + caused in vivo accumulation of trehalose-6P upon heat shock and prevented cell growth at 37 to 40°C. Accumulation of trehalose-6P in cells bearing a chromosomal disruption of the tpp1 + gene and containing a plasmid with tpp1 + under the control of the thiamine-repressible promotor correlated with tpp1 + repression. The level of tpp1 + mRNA rose upon heat shock, osmostress, or oxidative stress and was negatively controlled by cyclic AMP-dependent protein kinase activity. Expression of tpp1 + during oxidative or osmotic stress, but not during heat shock, was under positive control by the wis1-sty1 (equivalent to phh1 and spc1 ) mitogen-activated protein kinase pathway. Analysis of Tpp1 protein levels suggests that the synthesis of trehalose-6P phosphatase may also be subjected to translational or posttranslational control.

Recent studies have shown that heat shock proteins and trehalose synthesis are important factors in the thermotolerance of the fission yeast Schizosaccharomyces pombe . We examined the effects of trehalose-6-phosphate (trehalose-6P) synthase overexpression on resistance to several stresses in cells of S. pombe transformed with a plasmid bearing the tps1 gene, which codes for trehalose-6P synthase, under the control of the strong thiamine-repressible promoter. Upon induction of trehalose-6P synthase, the elevated levels of intracellular trehalose correlated not only with increased tolerance to heat shock but also with resistance to freezing and thawing, dehydration, osmostress, and toxic levels of ethanol, indicating that trehalose may be the stress metabolite underlying the overlap in induced tolerance to these stresses. Among the isogenic strains transformed with this construct, one in which the gene coding for the trehalose-hydrolyzing enzyme, neutral trehalase, was disrupted accumulated trehalose to a greater extent and was more resistant to the above stresses. Increased trehalose concentration is thus a major determinant of the general stress protection response in S. pombe .

We have cloned and sequenced the ntp1 + gene that codes for neutral trehalase in the fission yeast Schizosaccharomyces pombe . The ntp1 + gene product (Ntp1p) showed a 45–55% identity with neutral trehalases from other yeasts at the amino acid sequence level. However, in clear contrast to other neutral yeast trehalases so far characterized (which show two cAMP phospho-sites), only one consensus site for cAMP-dependent protein phosphorylation was found in Ntp1p. Northern blot hybridization experiments demonstrated that the Wis-Phh1/Sty1 MAP kinase cascade regulates ntp1 + expression during osmostress.

The fission yeast protein kinase C (PKC) ortholog Pck2 controls cell wall synthesis and is a major upstream activator of the cell integrity pathway (CIP) and its core component, the MAP kinase Pmk1 (also known as Spm1), in response to environmental stimuli. We show that in vivo phosphorylation of Pck2 at the conserved T842 activation loop during growth and in response to different stresses is mediated by the phosphoinositide-dependent kinase (PDK) ortholog Ksg1 and an autophosphorylation mechanism. However, T842 phosphorylation is not essential for Pmk1 activation, and putative phosphorylation at T846 might play an additional role in Pck2 catalytic activation and downstream signaling. These events, together with turn motif autophosphorylation at T984 and binding to small GTPases Rho1 and/or Rho2, stabilize Pck2 and render it competent to exert its biological functions. Remarkably, the target of rapamycin complex 2 (TORC2) does not participate in the catalytic activation of Pck2, but instead contributes to de novo Pck2 synthesis, which is essential to activate the CIP in response to cell wall damage or glucose exhaustion. These results unveil a novel mechanism whereby TOR regulates PKC function at a translational level, and they add a new regulatory layer to MAPK signaling cascades.

Soto, T., Fernandez, J., Vicente-Soler, J., Cansado, J., and Gacto, M. 1996. Posttranslational regulatory control of trehalase induced by nutrients, metabolic inhibitors, and physical agents in Pachysolen tannophilus. Fungal Genetics and Biology 20, 143–151. Trehalase activity in the yeast Pachysolen tannophilus is due to a single enzyme with highest activity during exponential growth on glucose. Derepressed cells enhanced markedly the level of trehalase activity upon addition of glucose, 2,4-dinitrophenol, nitrogen sources, protein-synthesis inhibitors, or azide. The increase induced by the last three types of compounds required the presence of an energy source in the incubation assay. In glucose-repressed cells, only nitrogen sources and azide were able to produce trehalase activation. The increase in trehalase was preceded by a cAMP signal, suggesting that cAMP may be involved as second messenger in a signaling pathway for trehalase stimulation. When cells from either repressed or derepressed growing cultures were temperature shifted from 28 to 40°C, trehalase activity increased with negligible change in the cAMP level. This type of activation was not produced in cells from resting cultures. The increase in trehalase triggered by heating was independent on both de novo protein synthesis and the presence of glucose. Treatment by phosphatase of trehalase activated by either glucose or heat shock resulted in trehalase deactivation, indicating that phosphorylation of the enzyme protein occurs during activation. These data support that at least two independent mechanisms, involving different phosphorylation pathways, can promote trehalase activation in Pa. tannophilus. Some functional features of these signaling pathways appear closer to those present in the fission yeast Schizosaccharomyces pombe than to those found in other budding yeasts like Saccharomyces cerevisiae or Candida utilis.

14 pags, 8 figs, 1 tab, Mitogen-activated protein kinase (MAPK) signaling pathways play a fundamental role in the response of eukaryotic cells to environmental changes. Also, much evidence shows that the stimulus-dependent nuclear targeting of this class of regulatory kinases is crucial for adequate regulation of distinct cellular events. In the fission yeast Schizosaccharomyces pombe, the cell integrity MAPK pathway, whose central element is the MAPK Pmk1, regulates multiple processes such as cell wall integrity, vacuole fusion, cytokinesis, and ionic homeostasis. In non-stressed cells Pmk1 is constitutively localized in both cytoplasm and nucleus, and its localization pattern appears unaffected by its activation status or in response to stress, thus questioning the biological significance of the presence of this MAPK into the nucleus. We have addressed this issue by characterizing mutants expressing Pmk1 versions excluded from the cell nucleus and anchored to the plasma membrane in different genetic backgrounds. Although nuclear Pmk1 partially regulates cell wall integrity at a transcriptional level, membrane-tethered Pmk1 performs many of the biological functions assigned to wild type MAPK like regulation of chloride homeostasis, vacuole fusion, and cellular separation. However, we found that down-regulation of nuclear Pmk1 by MAPK phosphatases induced by the stress activated protein kinase pathway is important for the fine modulation of extranuclear Pmk 1activity. These results highlight the importance of the control of MAPK activity at subcellular level. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc., This work was supported in part by Ministerio de Educación, Cultura y Deporte Grants BFU2011-22517 (to J. C.) and BFU2010-15641 (to P. P.) and 15280/PI/10 from Fundación Séneca, Spain.

Fission yeast mitogen-activated protein kinase (MAPK) Pmk1p is involved in morphogenesis, cytokinesis, and ion homeostasis as part of the cell integrity pathway, and it becomes activated under multiple stresses, including hyper- or hypotonic conditions, glucose deprivation, cell wall-damaging compounds, and oxidative stress. The only protein phosphatase known to dephosphorylate and inactivate Pmk1p is Pmp1p. We show here that the stress-activated protein kinase (SAPK) pathway and its main effector, Sty1p MAPK, are essential for proper deactivation of Pmk1p under hypertonic stress in a process regulated by Atf1p transcription factor. We demonstrate that tyrosine phosphatases Pyp1p and Pyp2p, and serine/threonine phosphatase Ptc1p, that negatively regulate Sty1p activity and whose expression is dependent on Sty1p-Atf1p function, are involved in Pmk1p dephosphorylation under osmostress. Pyp1p and Ptc1p, in addition to Pmp1p, also control the basal level of MAPK Pmk1p activity in growing cells and associate with, and dephosphorylate Pmk1p both in vitro and in vivo. Our results with Ptc1p provide the first biochemical evidence for a PP2C-type phosphatase acting on more than one MAPK in yeast cells. Importantly, the SAPK-dependent down-regulation of Pmk1p through Pyp1p, Pyp2p, and Ptc1p was not complete, and Pyp1p and Ptc1p phosphatases are able to negatively regulate MAPK Pmk1p activity by an alternative regulatory mechanism. Our data also indicate that Pmk1p phosphorylation oscillates as a function of the cell cycle, peaking at cell separation during cytokinesis, and that Pmp1p phosphatase plays a main role in regulating this process.

In synchronized light-dark cycles, stationary-phase cultures of the budding yeast Candida utilis were able to survive heat treatment at 50 degees C with an apparent circadian-like rhythm related to the onset of light. However, in continuous darkness this pattern did not run freely and was markedly dampened. We discuss these findings in terms of the potential circadian control of heat tolerance, which has been described in the fission yeast Schizosaccharomyces pombe. Our results suggest that the resistance pattern observed in C. utilis is most likely an adaptive response to the light-induced generation of reactive oxygen species rather than the occurrence of a truly endogenous circadian rhythm.

One intriguing challenge in modern biology is to understand how cells respond to, and distinguish between different stressing stimuli. Evidence accumulated in recent years indicates that a network of signaling pathways extends from the plasma membrane to the very core of the cell nucleus to transduce environmental changes into a graded transcriptional response. Although many steps still remain unclear, studies on the stress-activated protein kinase (SAPK) pathways and related mechanisms provide insight into the biochemistry that regulates signal transmission and leads to outcomes such as cell adaptation and differentiation. This review focuses on selected topics of current interest related to the sensing of stress signals in cells of the fission yeast Schizosaccharomyces pombe. Because signaling pathways appear to be evolutionarily well conserved, yeasts may be useful models to learn how higher eukaryotes sense and respond to stresses at the cellular level.

In the fission yeast Schizosaccharomyces pombe the Wak1p/Win1p-Wis1p-Sty1p stress-activated protein kinase (SAPK) pathway relays environmental signals to the transcriptional machinery and modulates gene expression via a cascade of protein phosphorylation. Cells of S. pombe subjected to cold shock (transfer from 28 degrees C to 15 degrees C) transiently activated the Sty1p mitogen-activated protein kinase (MAPK) by phosphorylation. Induction of this response was completely abolished in cells disrupted in the upstream response regulator Mcs4p. The cold-triggered Sty1p activation was partially dependent on Wak1p MAPKKK and fully dependent on Wis1p MAPKK suggesting that the signal transmission follows a branched pathway, with the redundant MAPKKK Win1p as alternative transducer to Wis1p, which subsequently activates the effector Sty1p MAPK. Also, the bZIP transcription factor Atf1p became phosphorylated in a Sty1p-dependent way during the cold shock and this phosphorylation was found responsible for the increased expression of gpd1+, ctt1+, tps1+ and ntp1+ genes. Strains deleted in transcription factors Atf1p or Pcr1p were unable to grow upon incubation at low temperature whereas those disrupted in any member of the SAPK pathway were able to do so. These data reveal that S. pombe responds to cold by inducing the SAPK pathway. However, such activation is dispensable for yeast growth in cold conditions, supporting that the presence of Atf1/Pcr1 heterodimers, rather than an operative SAPK pathway, is critical to ensure yeast growth at low temperature by an as yet undefined mechanism.

Trehalose metabolism is an essential component of the stress response in yeast cells. In this work we show that the products of the principal genes involved in trehalose metabolism in Schizosaccharomyces pombe, tps1+ (coding for trehalose-6-P synthase, Tps1p), ntp1+ (encoding neutral trehalase, Ntp1p) and tpp1+ (that codes for trehalose-6-P phosphatase, Tpp1p), interact in vitro with each other and with themselves to form protein complexes. Disruption of the gene tps1+ blocks the activation of the neutral trehalase induced by heat shock but not by osmotic stress. We propose that this association may reflect the Tps1p-dependent requirement for thermal activation of trehalase. Data reported here indicate that following a heat shock the enzyme activity of trehalase is associated with Ntp1p dimers or trimers but not with either Ntp1p monomers or with complexes involving Tps1p. These results raise the possibility that heat shock and osmotic stress activate trehalase differentially by acting in the first case through an specific mechanism involving Tps1p-Ntp1p complexes. This study provides the first evidence for the participation of the catabolic enzyme trehalase in the structural framework of a regulatory macromolecular complex containing trehalose-6-P synthase in the fission yeast.

Cells of Schizosaccharomyces pombe disrupted in the tps1+ gene, which encodes trehalose-6P synthase, were unable to increase trehalase activity in response to the addition of glucose or nitrogen source. Moreover, in contrast to normal cells, Deltatps1 cells did not increase trehalase activity by heat shock. Overexpression of tps1+ in cells devoid of trehalose-6P synthase restored the ability to increase trehalase after addition of nutrients or by heat shock. In glucose-repressed cells, which are normally refractory to the activation of trehalase by glucose, overexpression of tps1+ enabled the cells to increase trehalase activity upon addition of the sugar. Northern hybridisations were used to determine the level of mRNA for trehalase in normal and Deltatps1 cells. Transcription for trehalase was not significantly altered upon addition of glucose or nitrogen source, but increased markedly in heat-shocked cells even though trehalase activity remained unchanged in Deltatps1 cells. These findings provide evidence for a role of trehalose-6P synthase in the signalling pathway causing post-transcriptional activation of neutral trehalase induced by nutrients or heat shock. However, trehalase increased in Deltatps1 cells under hypertonic conditions suggesting the existence in Schiz. pombe of a distinct regulatory mechanism for enhancement of trehalase, specifically triggered by osmostress.

Exposure of cells of Schizosaccharomyces pombe to heat shock or osmotic upshift results in an increased level of neutral trehalase activity, which is responsible for hydrolysis of intracellular trehalose. We constructed S. pombe mutants lacking neutral trehalase activity by gene replacement at the newly defined ntp1 + locus. Analysis of these mutants revealed that a twofold increase in trehalose accumulation, enhanced acquired thermoresistance, and marked salt tolerance characterized their ability to grow in liquid and solid media. Analysis of the expression of the trehalase gene under heat shock and osmotic upshift revealed the transcriptional activation of ntp1 + in response to both stresses.

Background Glucose is a signaling molecule which regulates multiple events in eukaryotic organisms and the most preferred carbon source in the fission yeast Schizosaccharomyces pombe. The ability of this yeast to grow in the absence of glucose becomes strongly limited due to lack of enzymes of the glyoxylate cycle that support diauxic growth. The stress-activated protein kinase (SAPK) pathway and its effectors, Sty1 MAPK and transcription factor Atf1, play a critical role in the adaptation of fission yeast to grow on alternative non-fermentable carbon sources by inducing the expression of fbp1+ gene, coding for the gluconeogenic enzyme fructose-1,6-bisphosphatase. The cell integrity Pmk1 pathway is another MAPK cascade that regulates various processes in fission yeast, including cell wall construction, cytokinesis, and ionic homeostasis. Pmk1 pathway also becomes strongly activated in response to glucose deprivation but its role during glucose exhaustion and ensuing adaptation to respiratory metabolism is currently unknown. Results We found that Pmk1 activation in the absence of glucose takes place only after complete depletion of this carbon source and that such activation is not related to an endogenous oxidative stress. Notably, Pmk1 MAPK activation relies on de novo protein synthesis, is independent on known upstream activators of the pathway like Rho2 GTPase, and involves PKC ortholog Pck2. Also, the Glucose/cAMP pathway is required operative for full activation of the Pmk1 signaling cascade. Mutants lacking Pmk1 displayed a partial growth defect in respiratory media which was not observed in the presence of glucose. This phenotype was accompanied by a decreased and delayed expression of transcription factor Atf1 and target genes fbp1+ and pyp2+. Intriguingly, the kinetics of Sty1 activation in Pmk1-less cells was clearly altered during growth adaptation to non-fermentable carbon sources. Conclusions Unknown upstream elements mediate Pck2-dependent signal transduction of glucose withdrawal to the cell integrity MAPK pathway. This signaling cascade reinforces the adaptive response of fission yeast to such nutritional stress by enhancing the activity of the SAPK pathway.