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The Wis1p-Sty1p mitogen-activated protein kinase cascade is a major signalling system in the fission yeast Schizosaccharomyces pombe for a wide range of stress responses. It is known that trehalose functions as a protective metabolite to counteract deleterious effects of environmental stresses. Herein it is reported that the expression of genes related to trehalose metabolism in S. pombe, [ntp1.sup.+] (neutral trehalase) and [tps1.sup.+] [trehalose-6-phosphate (T6P) synthase], is partially regulated by the Sty1p kinase under salt-induced osmotic stress and conditions of slight oxidative stress and is fully dependent on this kinase under severe oxidative stress. This control is carried out through transcription factors Atf1p/Pcr1p during osmotic stress and through Pap1p during exposure to low levels of oxidative stress. However, all three transcription factors are needed for gene expression under conditions of extreme oxidative stress. In addition, a role for Sty1 p in the modulation of post-transcriptional activation of trehalase mediated by Pka1p/Sck1p kinases, as well as in the activity of T6P synthase under such stressful conditions has been demonstrated. These results reveal a novel dual action of the Wis1p-Sty1p pathway in the regulation of trehalose metabolism in fission yeast.

Disruptant strains of Schizosaccharomyces pombe that lack either Sck1, Pka1 or both protein kinases were used to assess the potential of trehalase as a common target for Sck1 and Pka1. Except in repressed cells, Sck1 was found involved in activation of trehalase. It was also found essential in the mechanism which is responsible for trehalase stimulation triggered by glucose and nitrogen source.

A cyclic-adenylic acid (cAMP) signal and cytoplasmic neutral trehalase activation in derepressed growing cells of Schizosaccharomyces pombe is observed by adding glucose. The trehalase activation requires a cAMP-dependent protein kinase (PKA1). During the presence of a cAMP-independent protein kinase, the activation occurs via a glucose- induced phosphorylation mechanism. Trehalase is the natural substrate for PKA1 and is a target for at least another protein kinase of the fission yeast.

A study of signaling pathways involved in the induction of cytoplasmic trehalase in Candida utilis reveals the participation of three different mechanisms that respond to diverse environmental or nutritional conditions to enhance trehalase activity. The cAMP-independent pathway is activated by protein synthesis and glucose and is functional only in repressed cells, while the glucose-stimulated pathway stimulates the target enzymes through some glucose-repressive element expression. A temporary cAMP enhancement and protein kinase activation are essential for the transduction of this signal.

Abstract 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.

Non-muscle myosin II activation by regulatory light chain (Rlc1 Sp ) phosphorylation at Ser35 is crucial for cytokinesis during respiration in the fission yeast Schizosaccharomyces pombe. We show that in the early divergent and dimorphic fission yeast S. japonicus non-phosphorylated Rlc1 Sj regulates the activity of Myo2 Sj and Myp2 Sj heavy chains during cytokinesis. Intriguingly, Rlc1 Sj -Myo2 Sj nodes delay yeast to hyphae onset but are essential for mycelial development. Structure-function analysis revealed that phosphorylation-induced folding of Rlc1 Sp α1 helix into an open conformation allows precise regulation of Myo2 Sp during cytokinesis. Consistently, inclusion of bulky tryptophan residues in the adjacent α5 helix triggered Rlc1 Sp shift and supported cytokinesis in absence of Ser35 phosphorylation. Remarkably, unphosphorylated Rlc1 Sj lacking the α1 helix was competent to regulate S. pombe cytokinesis during respiration. Hence, early diversification resulted in two efficient phosphorylation-independent and -dependent modes of Rlc1 regulation of myosin II activity in fission yeasts, the latter being conserved through evolution., Competing Interests: The authors declare no competing interests., (© 2023 Universidad de Murcia.)

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 Saccharomyces cerevisiae , little is known about how this coping response is regulated in the evolutionary distant fission yeast Schizosaccharomyces pombe . Here, we show that S. pombe autophagy in response to glucose limitation relies on mitochondrial respiration and the electron transport chain (ETC), but, in contrast to S. cerevisiae , the AMP-activated protein kinase (AMPK) and DNA damage response pathway components do not modulate fission yeast autophagic flux under these conditions. In the presence of glucose, the cAMP-protein kinase A (PKA) signaling pathway constitutively represses S. pombe autophagy by downregulating the transcription factor Rst2, which promotes the expression of respiratory genes required for autophagy induction under limited glucose availability. Furthermore, the stress-activated protein kinase (SAPK) signaling pathway, and its central mitogen-activated protein kinase (MAPK) Sty1, positively modulate autophagy upon glucose limitation at the transcriptional level through its downstream effector Atf1 and by direct in vivo phosphorylation of Rst2 at S292. Thus, our data indicate that the signaling pathways that govern autophagy during glucose shortage or starvation have evolved differently in S. pombe and uncover the existence of sophisticated and multifaceted mechanisms that control this self-preservation and survival response.

Cytokinesis, the separation of daughter cells at the end of mitosis, relies in animal cells on a contractile actomyosin ring (CAR) composed of actin and class II myosins, whose activity is strongly influenced by regulatory light chain (RLC) phosphorylation. However, in simple eukaryotes such as the fission yeast Schizosaccharomyces pombe , RLC phosphorylation appears dispensable for regulating CAR dynamics. We found that redundant phosphorylation at Ser35 of the S. pombe RLC homolog Rlc1 by the p21-activated kinases Pak1 and Pak2, modulates myosin II Myo2 activity and becomes essential for cytokinesis and cell growth during respiration. Previously, we showed that the stress-activated protein kinase pathway (SAPK) MAPK Sty1 controls fission yeast CAR integrity by downregulating formin For3 levels (Gómez-Gil et al., 2020). Here, we report that the reduced availability of formin For3-nucleated actin filaments for the CAR is the main reason for the required control of myosin II contractile activity by RLC phosphorylation during respiration-induced oxidative stress. Thus, the restoration of For3 levels by antioxidants overrides the control of myosin II function regulated by RLC phosphorylation, allowing cytokinesis and cell proliferation during respiration. Therefore, fine-tuned interplay between myosin II function through Rlc1 phosphorylation and environmentally controlled actin filament availability is critical for a successful cytokinesis in response to a switch to a respiratory carbohydrate metabolism., Competing Interests: FP, EG, RM, AP, JV, AF, TS, PP, MM, JC No competing interests declared, (© 2023, Prieto-Ruiz et al.)

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.

Mitogen activated protein kinase (MAPK) signaling pathways execute essential functions in eukaryotic organisms by transducing extracellular stimuli into adaptive cellular responses. In the fission yeast model Schizosaccharomyces pombe the cell integrity pathway (CIP) and its core effector, MAPK Pmk1, play a key role during regulation of cell integrity, cytokinesis, and ionic homeostasis. Schizosaccharomyces japonicus , another fission yeast species, shows remarkable differences with respect to S. pombe , including a robust yeast to hyphae dimorphism in response to environmental changes. We show that the CIP MAPK module architecture and its upstream regulators, PKC orthologs Pck1 and Pck2, are conserved in both fission yeast species. However, some of S. pombe 's CIP-related functions, such as cytokinetic control and response to glucose availability, are regulated differently in S. japonicus. Moreover, Pck1 and Pck2 antagonistically regulate S. japonicus hyphal differentiation through fine-tuning of Pmk1 activity. Chimeric MAPK-swapping experiments revealed that S. japonicus Pmk1 is fully functional in S. pombe , whereas S. pombe Pmk1 shows a limited ability to execute CIP functions and promote S. japonicus mycelial development. Our findings also suggest that a modified N-lobe domain secondary structure within S. japonicus Pmk1 has a major influence on the CIP signaling features of this evolutionarily diverged fission yeast.

The Rho family of GTPases represents highly conserved molecular switches involved in a plethora of physiological processes. Fission yeast Schizosaccharomyces pombe has become a fundamental model organism to study the functions of Rho GTPases over the past few decades. In recent years, another fission yeast species, Schizosaccharomyces japonicus , has come into focus offering insight into evolutionary changes within the genus. Both fission yeasts contain only six Rho-type GTPases that are spatiotemporally controlled by multiple guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), and whose intricate regulation in response to external cues is starting to be uncovered. In the present review, we will outline and discuss the current knowledge and recent advances on how the fission yeasts Rho family GTPases regulate essential physiological processes such as morphogenesis and polarity, cellular integrity, cytokinesis and cellular differentiation.

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., Competing Interests: EG, RM, JV, AF, BV, FP, TS, PP, MM, JC No competing interests declared, (© 2020, Gómez-Gil et al.)

RNA-binding proteins (RBPs) play a major role during control of mRNA localization, stability, and translation and are central to most cellular processes. In the fission yeast Schizosaccharomyces pombe , the multiple K homology (KH) domain RBP Rnc1 downregulates the activity of the cell integrity pathway (CIP) via stabilization of pmp1 + mRNA, which encodes the Pmp1 phosphatase that inactivates Pmk1, the mitogen-activated protein kinase (MAPK) component of this signaling cascade. However, Rnc1 likely regulates the half-life/stability of additional mRNAs. We show that Rnc1 downregulates the activity of Sty1, the MAPK of the stress-activated MAPK pathway (SAPK), during control of cell length at division and recovery in response to acute stress. Importantly, this control strictly depends on Rnc1's ability to bind mRNAs encoding activators (Wak1 MAPKKK, Wis1 MAPKK) and downregulators (Atf1 transcription factor, Pyp1 and Pyp2 phosphatases) of Sty1 phosphorylation through its KH domains. Moreover, Sty1 is responsible for Rnc1 phosphorylation in vivo at multiple phosphosites during growth and stress, and these modifications trigger Rnc1 for proper binding and destabilization of the above mRNA targets. Phosphorylation by Sty1 prompts Rnc1-dependent mRNA destabilization to negatively control SAPK signaling, thus revealing an additional feedback mechanism that allows precise tuning of MAPK activity during unperturbed cell growth and stress. IMPORTANCE Control of mRNA localization, stability, turnover, and translation by RNA-binding proteins (RBPs) influences essential processes in all eukaryotes, including signaling by mitogen-activated protein kinase (MAPK) pathways. We describe that in the fission yeast Schizosaccharomyces pombe the RBP Rnc1 negatively regulates cell length at division during unperturbed growth and recovery after acute stress by reducing the activity of the MAPK Sty1, which regulates cell growth and differentiation during environmental cues. This mechanism relies on Rnc1 binding to specific mRNAs encoding both enhancers and negative regulators of Sty1 activity. Remarkably, multiple phosphorylation of Rnc1 by Sty1 favors RBP binding and destabilization of the above mRNAs. Thus, posttranscriptional modulation of MAP kinase signaling by RNA-binding proteins emerges as a major regulatory mechanism that dictates the growth cycle and cellular adaptation in response to the changing environment in eukaryotic organisms., (Copyright © 2020 Prieto-Ruiz et al.)

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

In eukaryotic cells, the highly conserved Target of Rapamycin (TOR) and the Mitogen Activated Protein Kinase (MAPK) signaling pathways elicit adaptive responses to extra- and intracellular conditions by regulating essential cellular functions. However, the nature of the functional relationships between both pathways is not fully understood. In the fission yeast Schizosaccharomyces pombe the cell integrity MAPK pathway (CIP) regulates morphogenesis, cell wall structure and ionic homeostasis. We show that the Rab GTPase Ryh1, a TORC2 complex activator, cross-activates the CIP and its core member, the MAPK Pmk1, by two distinct mechanisms. The first one involves TORC2 and its downstream effector, Akt ortholog Gad8, which together with TORC1 target Psk1 increase protein levels of the PKC ortholog Pck2 during cell wall stress or glucose starvation. Also, Ryh1 activates Pmk1 in a TORC2-independent fashion by prompting plasma membrane trafficking and stabilization of upstream activators of the MAPK cascade, including PDK ortholog Ksg1 or Rho1 GEF Rgf1. Besides, stress-activated Pmk1 cross-inhibits Ryh1 signaling by decreasing the GTPase activation cycle, and this ensures cell growth during alterations in phosphoinositide metabolism. Our results reveal a highly intricate cross-regulatory relationship between both pathways that warrants adequate cell adaptation and survival in response to environmental changes.

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., (© 2015. Published by The Company of Biologists Ltd.)

The fission yeast small GTPase Rho2 regulates morphogenesis and is an upstream activator of the cell integrity pathway, whose key element, mitogen-activated protein kinase (MAPK) Pmk1, becomes activated by multiple environmental stimuli and controls several cellular functions. Here we demonstrate that farnesylated Rho2 becomes palmitoylated in vivo at cysteine-196 within its carboxyl end and that this modification allows its specific targeting to the plasma membrane. Unlike that of other palmitoylated and prenylated GTPases, the Rho2 control of morphogenesis and Pmk1 activity is strictly dependent upon plasma membrane localization and is not found in other cellular membranes. Indeed, artificial plasma membrane targeting bypassed the Rho2 need for palmitoylation in order to signal. Detailed functional analysis of Rho2 chimeras fused to the carboxyl end from the essential GTPase Rho1 showed that GTPase palmitoylation is partially dependent on the prenylation context and confirmed that Rho2 signaling is independent of Rho GTP dissociation inhibitor (GDI) function. We further demonstrate that Rho2 is an in vivo substrate for DHHC family acyltransferase Erf2 palmitoyltransferase. Remarkably, Rho3, another Erf2 target, negatively regulates Pmk1 activity in a Rho2-independent fashion, thus revealing the existence of cross talk whereby both GTPases antagonistically modulate the activity of this MAPK cascade.

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 Pmk1 activity. These results highlight the importance of the control of MAPK activity at subcellular level.

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.

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.