RESUMO
BACKGROUND: The yeast Komagataella phaffii has become a very popular host for heterologous protein expression, very often based on the use of the AOX1 promoter, which becomes activated when cells are grown with methanol as a carbon source. However, the use of methanol in industrial settings is not devoid of problems, and therefore, the search for alternative expression methods has become a priority in the last few years. RESULTS: We recently reported that moderate alkalinization of the medium triggers a fast and wide transcriptional response in K. phaffii. Here, we present the utilization of three alkaline pH-responsive promoters (pTSA1, pHSP12 and pPHO89) to drive the expression of a secreted phytase enzyme by simply shifting the pH of the medium to 8.0. These promoters offer a wide range of strengths, and the production of phytase could be modulated by adjusting the pH to specific values. The TSA1 and PHO89 promoters offered exquisite regulation, with virtually no enzyme production at acidic pH, while limitation of Pi in the medium further potentiated alkaline pH-driven phytase expression from the PHO89 promoter. An evolved strain based on this promoter was able to produce twice as much phytase as the reference pAOX1-based strain. Functional mapping of the TSA1 and HSP12 promoters suggests that both contain at least two alkaline pH-sensitive regulatory regions. CONCLUSIONS: Our work shows that the use of alkaline pH-regulatable promoters could be a useful alternative to methanol-based expression systems, offering advantages in terms of simplicity, safety and economy.
Assuntos
6-Fitase , Saccharomycetales , Pichia/metabolismo , Metanol/metabolismo , 6-Fitase/genética , 6-Fitase/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/metabolismoRESUMO
Coenzyme A (CoA) is an essential cofactor involved in a range of metabolic pathways including the activation of long-chain fatty acids for catabolism. Cells synthesize CoA de novo from vitamin B5 (pantothenate) via a pathway strongly conserved across prokaryotes and eukaryotes. In humans, it involves five enzymatic steps catalyzed by four enzymes: pantothenate kinase (PANK [isoforms 1-4]), 4'-phosphopantothenoylcysteine synthetase (PPCS), phosphopantothenoylcysteine decarboxylase (PPCDC), and CoA synthase (COASY). To date, inborn errors of metabolism associated with all of these genes, except PPCDC, have been described, two related to neurodegeneration with brain iron accumulation (NBIA), and one associated with a cardiac phenotype. This paper reports another defect in this pathway (detected in two sisters), associated with a fatal cardiac phenotype, caused by biallelic variants (p.Thr53Pro and p.Ala95Val) of PPCDC. PPCDC enzyme (EC 4.1.1.36) catalyzes the decarboxylation of 4'-phosphopantothenoylcysteine to 4'-phosphopantetheine in CoA biosynthesis. The variants p.Thr53Pro and p.Ala95Val affect residues highly conserved across different species; p.Thr53Pro is involved in the binding of flavin mononucleotide, and p.Ala95Val is likely a destabilizing mutation. Patient-derived fibroblasts showed an absence of PPCDC protein, and nearly 50% reductions in CoA levels. The cells showed clear energy deficiency problems, with defects in mitochondrial respiration, and mostly glycolytic ATP synthesis. Functional studies performed in yeast suggest these mutations to be functionally relevant. In summary, this work describes a new, ultra-rare, severe inborn error of metabolism due to pathogenic variants of PPCDC.
Assuntos
Carboxiliases , Cardiomiopatia Dilatada , Humanos , Carboxiliases/genética , Coenzima A/genética , Coração , Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: Adaptation to alkalinization of the medium in fungi involves an extensive remodeling of gene expression. Komagataella phaffii is an ascomycetous yeast that has become an organism widely used for heterologous protein expression. We explore here the transcriptional impact of moderate alkalinization in this yeast, in search of suitable novel promoters able to drive transcription in response to the pH signal. RESULTS: In spite of a minor effect on growth, shifting the cultures from pH 5.5 to 8.0 or 8.2 provokes significant changes in the mRNA levels of over 700 genes. Functional categories such as arginine and methionine biosynthesis, non-reductive iron uptake and phosphate metabolism are enriched in induced genes, whereas many genes encoding iron-sulfur proteins or members of the respirasome were repressed. We also show that alkalinization is accompanied by oxidative stress and we propose this circumstance as a common trigger of a subset of the observed changes. PHO89, encoding a Na+/Pi cotransporter, appears among the most potently induced genes by high pH. We demonstrate that this response is mainly based on two calcineurin-dependent response elements located in its promoter, thus indicating that alkalinization triggers a calcium-mediated signal in K. phaffii. CONCLUSIONS: This work defines in K. phaffii a subset of genes and diverse cellular pathways that are altered in response to moderate alkalinization of the medium, thus setting the basis for developing novel pH-controlled systems for heterologous protein expression in this fungus.
Assuntos
Ascomicetos , Saccharomycetales , Transcriptoma , Saccharomycetales/genética , Perfilação da Expressão Gênica , Ascomicetos/genéticaRESUMO
The Saccharomyces cerevisiae ENA1 gene, encoding a Na+-ATPase, responds transcriptionally to the alkalinization of the medium by means of a network of signals that involves the Rim101, the Snf1 and PKA kinases, and the calcineurin/Crz1 pathways. We show here that the ENA1 promoter also contains a consensus sequence, located at nt -553/-544, for the Stp1/2 transcription factors, the downstream components of the amino acid sensing SPS pathway. Mutation of this sequence or deletion of either STP1 or STP2 decreases the activity of a reporter containing this region in response to alkalinization as well as to changes in the amino acid composition in the medium. Expression driven from the entire ENA1 promoter was affected with similar potency by the deletion of PTR3, SSY5, or simultaneous deletion of STP1 and STP2 when cells were exposed to alkaline pH or moderate salt stress. However, it was not altered by the deletion of SSY1, encoding the amino acid sensor. In fact, functional mapping of the ENA1 promoter reveals a region spanning from nt -742 to -577 that enhances transcription, specifically in the absence of Ssy1. We also found that the basal and alkaline pH-induced expression from the HXT2, TRX2, and, particularly, SIT1 promoters was notably decreased in an stp1 stp2 deletion mutant, whereas the PHO84 and PHO89 gene reporters were unaffected. Our findings add a further layer of complexity to the regulation of ENA1 and suggest that the SPS pathway might participate in the regulation of a subset of alkali-inducible genes.
Assuntos
Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Aminoácidos/metabolismo , Regulação Fúngica da Expressão Gênica , ATPase Trocadora de Sódio-Potássio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The fact that overexpression of the yeast Ser/Thr protein phosphatase Ppz1 induces a dramatic halt in cell proliferation was known long ago, but only work in the last few years has provided insight into the molecular basis for this toxicity. Overexpression of Ppz1 causes abundant changes in gene expression and modifies the phosphorylation state of more than 150 proteins, including key signaling protein kinases such as Hog1 or Snf1. Diverse cellular processes are altered: halt in translation, failure to properly adapt to low glucose supply, acidification of the cytosol, or depletion of intracellular potassium content are a few examples. Therefore, the toxicity derived from an excess of Ppz1 appears to be multifactorial, the characteristic cell growth blockage thus arising from the combination of various altered processes. Notably, overexpression of the Ppz1 regulatory subunit Hal3 fully counteracts the toxic effects of the phosphatase, and this process involves intracellular relocation of the phosphatase to internal membranes.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ciclo Celular , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Type 1 Ser/Thr protein phosphatases are represented in all fungi by two enzymes, the ubiquitous PP1, with a conserved catalytic polypeptide (PP1c) and numerous regulatory subunits, and PPZ, with a C-terminal catalytic domain related to PP1c and a variable N-terminal extension. Current evidence indicates that, although PP1 and PPZ enzymes might share some cellular targets and regulatory subunits, their functions are quite separated, and they have individual regulation. We explored the structures of PP1c and PPZ across 57 fungal species to identify those features that (1) are distinctive among these enzymes and (2) have been preserved through evolution. PP1c enzymes are more conserved than PPZs. Still, we identified 26 residues in the PP1 and PPZ catalytic moieties that are specific for each kind of phosphatase. In some cases, these differences likely affect the distribution of charges in the surface of the protein. In many fungi, Hal3 is a specific inhibitor of the PPZ phosphatases, although the basis for the interaction of these proteins is still obscure. By in vivo co-purification of the catalytic domain of ScPpz1 and ScHal3, followed by chemical cross-linking and MS analysis, we identified a likely Hal3-interacting region in ScPpz1 characterized by two major and conserved differences, D566 and D615 in ScPpz1, which correspond to K210 and K259 in ScPP1c (Glc7). Functional analysis showed that changing D615 to K renders Ppz1 refractory to Hal3 inhibition. Since ScHal3 does not regulate Glc7 but it inhibits all fungal PPZ tested so far, this conserved D residue could be pivotal for the differential regulation of both enzymes in fungi.
Assuntos
Fosfoproteínas Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Catálise , Domínio Catalítico/fisiologia , Fenótipo , Proteína Fosfatase 1/metabolismoRESUMO
In Yarrowia lipolytica, expression of the genes encoding the enzymes of the N-acetylglucosamine (NAGA) utilization pathway (NAG genes) becomes independent of the presence of NAGA in a Ylnag5 mutant lacking NAGA kinase. We addressed the question of whether the altered transcription was due to a lack of kinase activity or to a moonlighting role of this protein. Glucosamine-6-phosphate deaminase (Nag1) activity was measured as a reporter of NAG genes expression. The NGT1 gene encoding the NAGA transporter was deleted, creating a Ylnag5 ngt1 strain. In glucose cultures of this strain, Nag1 activity was similar to that of the Ylnag5 strain, ruling out the possibility that NAGA derived from cell wall turnover could trigger the derepression. Heterologous NAGA kinases were expressed in a Ylnag5 strain. Among them, the protein from Arabidopsis thaliana did not restore kinase activity but lowered Nag1 activity 4-fold with respect to a control. Expression in the Ylnag5 strain of YlNag5 variants F320S or D214V with low kinase activity caused a repression similar to that of the wild-type protein. Together, these results indicate that YlNag5 behaves as a moonlighting protein. An RNA-seq analysis revealed that the Ylnag5 mutation had a limited transcriptomic effect besides derepression of the NAG genes.
Assuntos
Perfilação da Expressão Gênica/métodos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Yarrowia/crescimento & desenvolvimento , Arabidopsis/enzimologia , Arabidopsis/genética , Clonagem Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Mutação , Análise de Sequência de RNA , Yarrowia/enzimologia , Yarrowia/genéticaRESUMO
Ppz Ser/Thr protein phosphatases (PPases) are found only in fungi and have been proposed as potential antifungal targets. In Saccharomyces cerevisiae Ppz1 (ScPpz1) is involved in regulation of monovalent cation homeostasis. ScPpz1 is inhibited by two regulatory proteins, Hal3 and Vhs3, which have moonlighting properties, contributing to the formation of an unusual heterotrimeric PPC decarboxylase (PPCDC) complex crucial for CoA biosynthesis. Here we report the functional characterization of CnPpz1 (CNAG_03673) and two possible Hal3-like proteins, CnHal3a (CNAG_00909) and CnHal3b (CNAG_07348) from the pathogenic fungus Cryptococcus neoformans. Deletion of CnPpz1 or CnHal3b led to phenotypes unrelated to those observed in the equivalent S. cerevisiae mutants, and the CnHal3b-deficient strain was less virulent. CnPpz1 is a functional PPase and partially replaced endogenous ScPpz1. Both CnHal3a and CnHal3b interact with ScPpz1 and CnPpz1 in vitro but do not inhibit their phosphatase activity. Consistently, when expressed in S. cerevisiae, they poorly reproduced the Ppz1-regulatory properties of ScHal3. In contrast, both proteins were functional monogenic PPCDCs. The CnHal3b isoform was crystallized and, for the first time, the 3D-structure of a fungal PPCDC elucidated. Therefore, our work provides the foundations for understanding the regulation and functional role of the Ppz1-Hal3 system in this important pathogenic fungus.
Assuntos
Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/genética , Proteínas Fúngicas/genética , Fosfoproteínas Fosfatases/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Modelos Moleculares , Fenótipo , Fosfoproteínas Fosfatases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Cryptococcus neoformans is an encapsulated pathogenic yeast that can change the size of the cells during infection. In particular, this process can occur by enlarging the size of the capsule without modifying the size of the cell body, or by increasing the diameter of the cell body, which is normally accompanied by an increase of the capsule too. This last process leads to the formation of cells of an abnormal enlarged size denominated titan cells. Previous works characterized titan cell formation during pulmonary infection but research on this topic has been hampered due to the difficulty to obtain them in vitro. In this work, we describe in vitro conditions (low nutrient, serum supplemented medium at neutral pH) that promote the transition from regular to titan-like cells. Moreover, addition of azide and static incubation of the cultures in a CO2 enriched atmosphere favored cellular enlargement. This transition occurred at low cell densities, suggesting that the process was regulated by quorum sensing molecules and it was independent of the cryptococcal serotype/species. Transition to titan-like cell was impaired by pharmacological inhibition of PKC signaling pathway. Analysis of the gene expression profile during the transition to titan-like cells showed overexpression of enzymes involved in carbohydrate metabolism, as well as proteins from the coatomer complex, and related to iron metabolism. Indeed, we observed that iron limitation also induced the formation of titan cells. Our gene expression analysis also revealed other elements involved in titan cell formation, such as calnexin, whose absence resulted in appearance of abnormal large cells even in regular rich media. In summary, our work provides a new alternative method to investigate titan cell formation devoid the bioethical problems that involve animal experimentation.
Assuntos
Cryptococcus neoformans/citologia , Cryptococcus neoformans/patogenicidade , Animais , Criptococose/microbiologia , Cryptococcus neoformans/genética , Perfilação da Expressão Gênica , Genes Fúngicos , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fagocitose , Fenótipo , Percepção de Quorum , Células RAW 264.7 , Transdução de SinaisRESUMO
The Ppz enzymes are Ser/Thr protein phosphatases present only in fungi that are characterized by a highly conserved C-terminal catalytic region, related to PP1c phosphatases, and a more divergent N-terminal extension. In Saccharomyces cerevisiae, Ppz phosphatases are encoded by two paralog genes, PPZ1 and PPZ2. Ppz1 is the most toxic protein when overexpressed in budding yeast, halting cell proliferation, and this effect requires its phosphatase activity. We show here that, in spite of their conserved catalytic domain, Ppz2 was not toxic when tested under the same conditions as Ppz1, albeit Ppz2 levels were somewhat lower. Remarkably, a hybrid protein composed of the N-terminal extension of Ppz1 and the catalytic domain of Ppz2 was as toxic as Ppz1, even if its expression level was comparable to that of Ppz2. Similar amounts of yeast PP1c (Glc7) produced an intermediate effect on growth. Mutation of the Ppz1 myristoylable Gly2 to Ala avoided the localization of the phosphatase at the cell periphery but only slightly attenuated its toxicity. Therefore, the N-terminal extension of Ppz1 plays a key role in defining Ppz1 toxicity. This region is predicted to be intrinsically disordered and contains several putative folding-upon-binding regions which are absent in Ppz2 and might be relevant for toxicity.
Assuntos
Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/toxicidade , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/toxicidade , Saccharomyces cerevisiae/metabolismo , Temperatura Alta , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Fenótipo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Dobramento de Proteína , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-AtividadeRESUMO
Maintenance of proper intracellular concentrations of monovalent cations, mainly sodium and potassium, is a requirement for survival of any cell. In the budding yeast Saccharomyces cerevisiae, monovalent cation homeostasis is determined by the active extrusion of protons through the Pma1 H+ -ATPase (reviewed in another chapter of this issue), the influx and efflux of these cations through the plasma membrane transporters (reviewed in this chapter), and the sequestration of toxic cations into the vacuoles. Here, we will describe the structure, function, and regulation of the plasma membrane transporters Trk1, Trk2, Tok1, Nha1, and Ena1, which play a key role in maintaining physiological intracellular concentrations of Na+ , K+ , and H+ , both under normal growth conditions and in response to stress.
Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cátions Monovalentes/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Homeostase , Transporte de Íons , Potássio/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , ATPases Translocadoras de Prótons , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Ppz enzymes are type-1 related Ser/Thr protein phosphatases that are restricted to fungi. In S. cerevisiae and other fungi, Ppz1 is involved in cation homeostasis and is regulated by two structurally-related inhibitory subunits, Hal3 and Vhs3, with Hal3 being the most physiologically relevant. Remarkably, Hal3 and Vhs3 have moonlighting properties, as they participate in an atypical heterotrimeric phosphopantothenoyl cysteine decarboxylase (PPCDC), a key enzyme for Coenzyme A biosynthesis. Here we identify and functionally characterize Ppz1 phosphatase (UmPpz1) and its presumed regulatory subunit (UmHal3) in the plant pathogen fungus Ustilago maydis. UmPpz1 is not an essential protein in U. maydis and, although possibly related to the cell wall integrity pathway, is not involved in monovalent cation homeostasis. The expression of UmPpz1 in S. cerevisiae Ppz1-deficient cells partially mimics the functions of the endogenous enzyme. In contrast to what was found in C. albicans and A. fumigatus, UmPpz1 is not a virulence determinant. UmHal3, an unusually large protein, is the only functional PPCDC in U. maydis and, therefore, an essential protein. However, when overexpressed in U. maydis or S. cerevisiae, UmHal3 does not reproduce Ppz1-inhibitory phenotypes. Indeed, UmHal3 does not inhibit UmPpz1 in vitro (although ScHal3 does). Therefore, UmHal3 might not be a moonlighting protein.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Fosfoproteínas Fosfatases/genética , Ustilago/fisiologia , Fenótipo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Doenças das Plantas/microbiologia , Proteínas Recombinantes , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de SequênciaRESUMO
Metal ions are essential elements for all living organisms. However, metals can be toxic when present in excess. In plants, metal homeostasis is partly achieved through the function of metal transporters, including the diverse natural resistance-associated macrophage proteins (NRAMP). Among them, the OsNramp6 gene encodes a previously uncharacterized member of the rice NRAMP family that undergoes alternative splicing to produce different NRAMP6 proteins. In this work, we determined the metal transport activity and biological role of the full-length and the shortest NRAMP6 proteins (l-NRAMP6 and s-NRAMP6, respectively). Both l-NRAMP6 and s-NRAMP6 are plasma membrane-localized proteins that function as iron and manganese transporters. The expression of l-Nramp6 and s-Nramp6 is regulated during infection with the fungal pathogen Magnaporthe oryzae, albeit with different kinetics. Rice plants grown under high iron supply show stronger induction of rice defense genes and enhanced resistance to M. oryzae infection. Also, loss of function of OsNramp6 results in enhanced resistance to M. oryzae, supporting the idea that OsNramp6 negatively regulates rice immunity. Furthermore, nramp6 plants showed reduced biomass, pointing to a role of OsNramp6 in plant growth. A better understanding of OsNramp6-mediated mechanisms underlying disease resistance in rice will help in developing appropriate strategies for crop protection.
Assuntos
Resistência à Doença , Ferro/metabolismo , Manganês/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Oryza/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Biomassa , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Teste de Complementação Genética , Magnaporthe/fisiologia , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Mutação/genética , Oryza/genética , Oryza/crescimento & desenvolvimento , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/metabolismoRESUMO
Lack of the yeast Ptc1 Ser/Thr protein phosphatase results in numerous phenotypic defects. A parallel search for high-copy number suppressors of three of these phenotypes (sensitivity to Calcofluor White, rapamycin and alkaline pH), allowed the isolation of 25 suppressor genes, which could be assigned to three main functional categories: maintenance of cell wall integrity (CWI), vacuolar function and protein sorting, and cell cycle regulation. The characterization of these genetic interactions strengthens the relevant role of Ptc1 in downregulating the Slt2-mediated CWI pathway. We show that under stress conditions activating the CWI pathway the ptc1 mutant displays hyperphosphorylated Cdc28 kinase and that these cells accumulate with duplicated DNA content, indicative of a G2-M arrest. Clb2-associated Cdc28 activity was also reduced in ptc1 cells. These alterations are attenuated by mutation of the MKK1 gene, encoding a MAP kinase kinase upstream Slt2. Therefore, our data show that Ptc1 is required for proper G2-M cell cycle transition after activation of the CWI pathway.
Assuntos
Proteína Fosfatase 2/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Parede Celular/genética , Parede Celular/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteína Fosfatase 2/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Polyphosphate (polyP) is a linear chain of up to hundreds of inorganic phosphate residues that is necessary for many physiological functions in all living organisms. In some bacteria, polyP supplies material to molecules such as DNA, thus playing an important role in biosynthetic processes in prokaryotes. In the present study, we set out to gain further insight into the role of polyP in eukaryotic cells. We observed that polyP amounts are cyclically regulated in Saccharomyces cerevisiae, and those mutants that cannot synthesise (vtc4Δ) or hydrolyse polyP (ppn1Δ, ppx1Δ) present impaired cell cycle progression. Further analysis revealed that polyP mutants show delayed nucleotide production and increased genomic instability. Based on these findings, we concluded that polyP not only maintains intracellular phosphate concentrations in response to fluctuations in extracellular phosphate levels, but also muffles internal cyclic phosphate fluctuations, such as those produced by the sudden demand of phosphate to synthetize deoxynucleotides just before and during DNA duplication. We propose that the presence of polyP in eukaryotic cells is required for the timely and accurate duplication of DNA.
Assuntos
Instabilidade Genômica , Polifosfatos/metabolismo , Saccharomyces cerevisiae/metabolismo , Pontos de Checagem do Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Organelas/metabolismo , Células Procarióticas/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genéticaRESUMO
Environmental alkalinisation represents a stress condition for yeast Saccharomyces cerevisiae, to which this organism responds with extensive gene expression remodelling. We show here that alkaline pH causes an overall decrease in the transcription rate (TR) and a fast destabilisation of mRNAs, followed by a more prolonged stabilisation phase. In many cases, augmented mRNA levels occur without the TR increasing, which can be attributed to mRNA stabilisation. In contrast, the reduced amount of mRNAs is contributed by both a drop in the TR and mRNA stability. A comparative analysis with other forms of stress shows that, unlike high pH stress, heat-shock, osmotic and oxidative stresses present a common transient increase in the TR. An analysis of environmentally-responsive (ESR) genes for the four above stresses suggests that up-regulated genes are governed mostly by TR changes and complex transient bidirectional changes in mRNA stability, whereas the down-regulated ESR gene set is driven by mRNA destabilisation and a lowered TR. In all the studied forms of stress, mRNA stability plays an important role in ESR. Overall, changes in mRNA levels do not closely reflect the rapid changes in the TR and stability upon exposure to stress, which highlights the existence of compensatory mechanisms.
Assuntos
Regulação Fúngica da Expressão Gênica/genética , RNA Mensageiro/biossíntese , Proteínas de Saccharomyces cerevisiae/biossíntese , Estresse Fisiológico/genética , Interação Gene-Ambiente , Concentração de Íons de Hidrogênio , Processamento Pós-Transcricional do RNA/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
BACKGROUND: Exposure of the budding Saccharomyces cerevisiae to an alkaline environment produces a robust transcriptional response involving hundreds of genes. Part of this response is triggered by an almost immediate burst of calcium that activates the Ser/Thr protein phosphatase calcineurin. Activated calcineurin dephosphorylates the transcription factor (TF) Crz1, which moves to the nucleus and binds to calcineurin/Crz1 responsive gene promoters. In this work we present a genome-wide study of the binding of Crz1 to gene promoters in response to high pH stress. RESULTS: Environmental alkalinization promoted a time-dependent recruitment of Crz1 to 152 intergenic regions, the vast majority between 1 and 5 min upon stress onset. Positional evaluation of the genomic coordinates combined with existing transcriptional studies allowed identifying 140 genes likely responsive to Crz1 regulation. Gene Ontology analysis confirmed the relevant impact of calcineurin/Crz1 on a set of genes involved in glucose utilization, and uncovered novel targets, such as genes responsible for trehalose metabolism. We also identified over a dozen of genes encoding TFs that are likely under the control of Crz1, suggesting a possible mechanism for amplification of the signal at the transcription level. Further analysis of the binding sites allowed refining the consensus sequence for Crz1 binding to gene promoters and the effect of chromatin accessibility in the timing of Crz1 recruitment to promoters. CONCLUSIONS: The present work defines at the genomic-wide level the kinetics of binding of Crz1 to gene promoters in response to alkaline stress, confirms diverse previously known Crz1 targets and identifies many putative novel ones. Because of the relevance of calcineurin/Crz1 in signaling diverse stress conditions, our data will contribute to understand the transcriptional response in other circumstances that also involve calcium signaling, such as exposition to sexual pheromones or saline stress.
Assuntos
Calcineurina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Redes Reguladoras de Genes , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/metabolismo , Regulação Fúngica da Expressão Gênica , Concentração de Íons de Hidrogênio , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Análise de Sequência de DNA/métodos , Transdução de Sinais , Estresse FisiológicoRESUMO
Maintenance of ion homeostatic mechanisms is essential for living cells, including the budding yeast Saccharomyces cerevisiae. Whereas the impact of changes in phosphate metabolism on metal ion homeostasis has been recently examined, the inverse effect is still largely unexplored. We show here that depletion of potassium from the medium or alteration of diverse regulatory pathways controlling potassium uptake, such as the Trk potassium transporters or the Pma1 H(+) -ATPase, triggers a response that mimics that of phosphate (Pi) deprivation, exemplified by accumulation of the high-affinity Pi transporter Pho84. This response is mediated by and requires the integrity of the PHO signaling pathway. Removal of potassium from the medium does not alter the amount of total or free intracellular Pi, but is accompanied by decreased ATP and ADP levels and rapid depletion of cellular polyphosphates. Therefore, our data do not support the notion of Pi being the major signaling molecule triggering phosphate-starvation responses. We also observe that cells with compromised potassium uptake cannot grow under limiting Pi conditions. The link between potassium and phosphate homeostasis reported here could explain the invasive phenotype, characteristic of nutrient deprivation, observed in potassium-deficient yeast cells.
Assuntos
Homeostase , Fosfatos/metabolismo , Potássio/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Citoplasma/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Polifosfatos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
Maintenance of appropriate fluxes of monovalent cation is a requirement for growth and survival. In the budding yeast Saccharomyces cerevisiae an electrochemical gradient of H(+) is fundamental for the uptake of diverse cations, such as K(+), and of many other nutrients. In spite of early work suggesting that alterations in monovalent cation fluxes impact on the uptake and utilization of nutrients, such as phosphate anions, only recently this important aspect of the yeast physiology has been addressed and characterized in some detail. This chapter provides a historical background and summarizes the latest findings.