RESUMO
The fission yeast Schizosaccharomyces pombe is an excellent model organism to explore cellular events owing to rich tools in genetics, molecular biology, cellular biology, and biochemistry. Schizosaccharomyces pombe proliferates continuously when nutrients are abundant but arrests in G1 phase upon depletion of nutrients such as nitrogen and glucose. When cells of opposite mating types are present, cells conjugate, fuse, undergo meiosis, and finally form 4 spores. This sexual differentiation process in S. pombe has been studied extensively. To execute sexual differentiation, the glucose-sensing cAMP-PKA (cyclic adenosine monophosphate-protein kinase A) pathway, nitrogen-sensing TOR (target of rapamycin) pathway, and SAPK (stress-activating protein kinase) pathway are crucial, and the MAPK (mitogen-activating protein kinase) cascade is essential for pheromone sensing. These signals regulate ste11 at the transcriptional and translational levels, and Ste11 is modified in multiple ways. This review summarizes the initiation of sexual differentiation in S. pombe based on results I have helped to obtain, including the work of many excellent researchers.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Fatores de Transcrição , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Regulação Fúngica da Expressão Gênica , Transdução de Sinais , Meiose , Feromônios/metabolismo , Diferenciação Sexual/genética , Glucose/metabolismo , Nitrogênio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/genética , Esporos Fúngicos/fisiologiaRESUMO
The phospholipase B homolog Plb1 and the cAMP-dependent protein kinase (PKA) pathway are required by fission yeast, also known as to Schizosaccharomyces pombe, to grow under KCl-stress conditions. Here, we report the relative contributions of Plb1 and the cAMP/PKA pathway during the hypertonic stress response. We show that the plb1∆, cyr1∆, and pka1∆ single mutants are sensitive to high concentrations of KCl but insensitive to sorbitol-induced osmotic stress. In contrast, the plb1∆ cyr1∆ and plb1∆ pka1∆ double mutants are hypersensitive to KCl and sorbitol. The cyr1∆ pka1∆ double mutants showed the same phenotype of each single mutant. Growth inhibition due to hypertonic stress in the plb1∆, plb1∆ cyr1∆, and plb1∆ pka1∆ strains was partially rescued by cgs1 deletion-cgs1∆ has constitutively active Pka1-or by the deletion of transcription factor Rst2, which is negatively regulated by Pka1. Pka1-GFP localized in the nucleus and cytoplasm in plb1∆, whereas it is localized only in the cytoplasm in cyr1∆, indicating that Plb1 does not regulate Pka1 localization. Glucose limitation downregulates the PKA pathway, and it was accordingly observed that glucose limitation in plb1∆ further increased the strain's sensitivity to KCl. Growth inhibition by KCl in plb1∆ under glucose-limited conditions was significantly rescued by cgs1∆ and slightly rescued by rst2∆. These findings indicate that, in fission yeast, Plb1 and the glucose-sensing cAMP/PKA pathway play a synergistic role in responding to hypertonic stress.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Pressão Osmótica , Lisofosfolipase/metabolismo , Glucose/metabolismo , Sorbitol/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Prenyldiphosphate synthases catalyze the reaction of allylic diphosphates with one or more isopentenyl diphosphate molecules to form compounds such as farnesyl diphosphate, used in, e.g., sterol biosynthesis and protein prenylation, as well as longer "polyprenyl" diphosphates, used in ubiquinone and menaquinone biosynthesis. Quinones play an essential role in electron transport and are associated with the inner mitochondrial membrane due to the presence of the polyprenyl group. In this work, we investigated the synthesis of the polyprenyl diphosphate that alkylates the ubiquinone ring precursor in Toxoplasma gondii, an opportunistic pathogen that causes serious disease in immunocompromised patients and the unborn fetus. The enzyme that catalyzes this early step of the ubiquinone synthesis is Coq1 (TgCoq1), and we show that it produces the C35 species heptaprenyl diphosphate. TgCoq1 localizes to the mitochondrion and is essential for in vitro T. gondii growth. We demonstrate that the growth defect of a T. gondii TgCoq1 mutant is rescued by complementation with a homologous TgCoq1 gene or with a (C45) solanesyl diphosphate synthase from Trypanosoma cruzi (TcSPPS). We find that a lipophilic bisphosphonate (BPH-1218) inhibits T. gondii growth at low-nanomolar concentrations, while overexpression of the TgCoq1 enzyme dramatically reduced growth inhibition by the bisphosphonate. Both the severe growth defect of the mutant and the inhibition by BPH-1218 were rescued by supplementation with a long-chain (C30) ubiquinone (UQ6). Importantly, BPH-1218 also protected mice against a lethal T. gondii infection. TgCoq1 thus represents a potential drug target that could be exploited for improved chemotherapy of toxoplasmosis. IMPORTANCE Millions of people are infected with Toxoplasma gondii, and the available treatment for toxoplasmosis is not ideal. Most of the drugs currently used are only effective for the acute infection, and treatment can trigger serious side effects requiring changes in the therapeutic approach. There is, therefore, a compelling need for safe and effective treatments for toxoplasmosis. In this work, we characterize an enzyme of the mitochondrion of T. gondii that can be inhibited by an isoprenoid pathway inhibitor. We present evidence that demonstrates that inhibition of the enzyme is linked to parasite death. In addition, the inhibitor can protect mice against a lethal dose of T. gondii. Our results thus reveal a promising chemotherapeutic target for the development of new medicines for toxoplasmosis.
Assuntos
Toxoplasma , Toxoplasmose , Animais , Camundongos , Difosfatos/metabolismo , Difosfonatos/farmacologia , Difosfonatos/uso terapêutico , Esteróis , Toxoplasmose/tratamento farmacológico , Toxoplasmose/prevenção & controle , Ubiquinona , Vitamina K 2/farmacologiaRESUMO
The cAMP-dependent protein kinase (Pka1) regulates many cellular events, including sexual development and glycogenesis, and response to the limitation of glucose, in Schizosaccharomyces pombe. Despite its importance in many cellular events, the targets of the cAMP/PKA pathway have not been fully investigated. Here, we demonstrate that the expression of mug14 is induced by downregulation of the cAMP/PKA pathway and limitation of glucose. This regulation is dependent on the function of Rst2, a transcription factor that regulates transition from mitosis to meiosis. The loss of the C2H2-type zinc finger domain in Rst2, termed Rst2 (C2H2∆), abolished the induction of Mug14 expression. Upon deletion of the stress starvation response element of the S. pombe (STREP: CCCCTC) sequence, which is a potential binding site of Rst2 on mug14, in the pka1∆ strain, its induction was abolished. The expression of Mug14 was significantly reduced and delayed by the limitation of glucose and also by nitrogen starvation in the rst2∆ strain. Mug14 is known to share a common function with Mde1 and Mta3 in the methionine salvage pathway, but the expression of mde1 and mta3 mRNAs was not enhanced by pka1 deletion and limitation of glucose. We conclude that the expression of Mug14 is upregulated by Rst2 under the control of the cAMP/PKA signaling pathway, which senses the limitation of glucose.
Assuntos
Proteínas de Ciclo Celular/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Fatores de Transcrição/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Glucose/metabolismo , Proteínas de Fluorescência Verde/genética , Sistema de Sinalização das MAP Quinases , Nitrogênio/metabolismo , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Fúngico , RNA Mensageiro , Proteínas Recombinantes de Fusão/genética , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Estresse FisiológicoRESUMO
Coenzyme Q (CoQ, ubiquinone) is an essential component of the electron transport system in aerobic organisms. Human type CoQ10, which has 10 units of isoprene in its quinone structure, is especially valuable as a food supplement. Therefore, studying the biosynthesis of CoQ10 is important not only for increasing metabolic knowledge, but also for improving biotechnological production. Herein, we show that Schizosaccharomyces pombe utilizes p-aminobenzoate (PABA) in addition to p-hydroxybenzoate (PHB) as a precursor for CoQ10 synthesis. We explored compounds that affect the synthesis of CoQ10 and found benzoic acid (Bz) at >5 µg/mL inhibited CoQ biosynthesis without accumulation of apparent CoQ intermediates. This inhibition was counteracted by incubation with a 10-fold lower amount of PABA or PHB. Overexpression of PHB-polyprenyl transferase encoded by ppt1 (coq2) also overcame the inhibition of CoQ biosynthesis by Bz. Inhibition by Bz was efficient in S. pombe and Schizosaccharomyces japonicus, but less so in Saccharomyces cerevisiae, Aureobasidium pullulans, and Escherichia coli. Bz also inhibited a S. pombe ppt1 (coq2) deletion strain expressing human COQ2, and this strain also utilized PABA as a precursor of CoQ10. Thus, Bz is likely to inhibit prenylation reactions involving PHB or PABA catalyzed by Coq2.
Assuntos
Ácido Benzoico/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas de Schizosaccharomyces pombe/biossíntese , Schizosaccharomyces/metabolismo , Ubiquinona/biossíntese , Proibitinas , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Ubiquinona/genéticaRESUMO
The cAMP-dependent protein kinase Pka1 is known as a regulator of glycogenesis, transition into meiosis, proper chromosome segregation, and stress responses in Schizosaccharomyces pombe. We demonstrated that both the cAMP/PKA pathway and glucose limitation play roles in appropriate spindle formation. Overexpression of Mal3 (1-308), an EB1 family protein, caused growth defects, increased 4C DNA content, and induced monopolar spindle formation. Overproduction of a high-affinity microtubule binding mutant (Q89R) and a recombinant protein possessing the CH and EB1 domains (1-241) both resulted in more severe phenotypes than Mal3 (1-308). Loss of functional Pka1 and glucose limitation rescued the phenotypes of Mal3-overexpressing cells, whereas deletion of Tor1 or Ssp2 did not. Growth defects and monopolar spindle formation in a kinesin-5 mutant, cut7-446, was partially rescued by pka1 deletion or glucose limitation. These findings suggest that Pka1 and glucose limitation regulate proper spindle formation in Mal3-overexpressing cells and the cut7-446 mutant.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Regulação Fúngica da Expressão Gênica , Glucose/deficiência , Cinesinas/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Fuso Acromático/metabolismo , Substituição de Aminoácidos , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/deficiência , DNA Fúngico/genética , DNA Fúngico/metabolismo , Deleção de Genes , Glucose/farmacologia , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Mutação , Fenótipo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/ultraestruturaRESUMO
The cAMP-dependent protein kinase Pka1 is known as a regulator of glycogenesis, transition into meiosis, chronological aging, and stress responses in the fission yeast, Schizosaccharomyces pombe. We demonstrated here that Pka1 is responsible for normal growth in the presence of the microtubule-destabilization drug TBZ and proper chromosome segregation. The deletion of the pka1 gene resulted in the TBZ-sensitive phenotype and chromosome mis-segregation. We isolated the mal3 gene as a multi-copy suppressor of the TBZ-sensitive phenotype in the pka1Δ strains. Overexpression of the CH domain (1-143) or the high-affinity microtubule binding mutant (1-143 Q89R) of Mal3 rescued the TBZ-sensitive phenotype in the pka1Δ and mal3Δ strains, while the EB1 domain (135-308) and the mutants defective in microtubule binding (1-143 Q89E) failed to do so in the same strains. Chromosome mis-segregation caused by TBZ in the pka1Δ or mal3Δ strains was suppressed by the overexpression of the Mal3 CH domain (1-143), Mal3 CH domain with the coiled-coil domain (1-197), or full-length Mal3. Overexpression of EB1 orthologs from Saccharomyces cerevisiae, Arabidopsis thaliana, Mus musculus, or Homo sapiens suppressed the TBZ-sensitive phenotype in the pka1Δ strains, indicating their conserved functions. These findings suggest that Pka1 and the microtubule binding of the Mal3 CH domain play a role in the maintenance of proper chromosome segregation.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Animais , Segregação de Cromossomos , Genes Fúngicos , Genes Supressores , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mutação , Fenótipo , Domínios Proteicos , Schizosaccharomyces/efeitos dos fármacos , Proteínas de Schizosaccharomyces pombe/química , Moduladores de Tubulina/farmacologiaRESUMO
Coenzyme Q (CoQ) is an essential component of the electron transport system that produces ATP in nearly all living cells. CoQ10 is a popular commercial food supplement around the world, and demand for efficient production of this molecule has increased in recent years. In this study, we explored CoQ10 production in the fission yeast Schizosaccharomyces pombe. We found that CoQ10 level was higher in stationary phase than in log phase, and that it increased when the cells were grown in a low concentration of glucose, in maltose, or in glycerol/ethanol medium. Because glucose signaling is mediated by cAMP, we evaluated the involvement of this pathway in CoQ biosynthesis. Loss of Pka1, the catalytic subunit of cAMP-dependent protein kinase, increased production of CoQ10, whereas loss of the regulatory subunit Cgs1 decreased production. Manipulation of other components of the cAMP-signaling pathway affected CoQ10 production in a consistent manner. We also found that glycerol metabolism was controlled by the cAMP/PKA pathway. CoQ10 production by the S. pombe ∆pka1 reached 0.98 mg/g dry cell weight in medium containing a non-fermentable carbon source [2% glycerol (w/v) and 1% ethanol (w/v) supplemented with 0.5% casamino acids (w/v)], twofold higher than the production in wild-type cells under normal growth conditions. These findings demonstrate that carbon source, growth phase, and the cAMP-signaling pathway are important factors in CoQ10 production in S. pombe.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Fúngicas/genética , Glucose/química , Schizosaccharomyces/metabolismo , Ubiquinona/análogos & derivados , Domínio Catalítico , Meios de Cultura/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Deleção de Genes , Maltose/química , Transdução de Sinais , Ubiquinona/biossíntese , Ubiquinona/metabolismoRESUMO
In Schizosaccharomyces pombe, the transcription factor Rst2 regulates ste11 in meiosis and fbp1 in glucogenesis downstream of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) pathway. Here, we demonstrate that Rst2 regulates additional cellular events. Overexpressed Rst2 elevated the frequency of oval, bent, branched, septated, and multi-septated cells. Cells showed normal nuclear divisions but exhibited abnormal nuclear organization at low frequency. In oval cells, microtubules were curved but they were rescued by the deletion of mal3. Since growth defect was not rescued by mal3 deletion, we argue that it is regulated independently. Loss of functional Pka1 exaggerated growth defect upon Rst2 overexpression because its downregulation by Pka1 was lost. Overexpression of Rst2 also caused sensitivity to KCl and CaCl2. These findings suggest that, in addition to meiosis and glucogenesis, Rst2 is involved in cellular events such as regulation of cell growth, cell morphology, mitosis progression, microtubules structure, nuclear structure, and stress response.
Assuntos
Microtúbulos/metabolismo , Mitose/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Fatores de Transcrição/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citocinese/efeitos dos fármacos , Citocinese/genética , Expressão Gênica , Meiose/efeitos dos fármacos , Meiose/genética , Microtúbulos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Fenótipo , Sais/farmacologia , Schizosaccharomyces/efeitos dos fármacos , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Cell lysis is induced in Schizosaccharomyces pombe ∆ura4 cells grown in YPD medium, which contains yeast extract, polypeptone, and glucose. To identify the medium components that induce cell lysis, we first tested various kinds of yeast extracts from different suppliers. Cell lysis of ∆ura4 cells on YE medium was observed when yeast extracts from OXOID, BD, Oriental, and Difco were used, but not when using yeast extract from Kyokuto. To determine which compounds induced cell lysis, we subjected yeast extract and polypeptone to GC-MS analysis. Ten kinds of compounds were detected in OXOID and BD yeast extracts, but not in Kyokuto yeast extract. Among them was urea, which was also present in polypeptone, and it clearly induced cell lysis. Deletion of the ure2 gene, which is responsible for utilizing urea, abolished the lytic effect of urea. The effect of urea was suppressed by deletion of pub1, and a similar phenotype was observed in the presence of polypeptone. Thus, urea is an inducer of cell lysis in S. pombe ∆ura4 cells.
Assuntos
Deleção de Genes , Regulação Fúngica da Expressão Gênica , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/efeitos dos fármacos , Ureia/toxicidade , Carbono-Nitrogênio Ligases/deficiência , Carbono-Nitrogênio Ligases/genética , Misturas Complexas/química , Misturas Complexas/farmacologia , Meios de Cultura , Cromatografia Gasosa-Espectrometria de Massas , Glucose/metabolismo , Glucose/farmacologia , Peptonas/metabolismo , Peptonas/farmacologia , Saccharomyces cerevisiae/química , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Coenzyme Q (CoQ) is composed of a benzoquinone moiety and an isoprenoid side chain of varying lengths. The length of the side chain is controlled by polyprenyl diphosphate synthase. In this study, dps1 genes encoding decaprenyl diphosphate synthase were cloned from three fungi: Bulleromyces albus, Saitoella complicata, and Rhodotorula minuta. The predicted Dps1 proteins contained seven conserved domains found in typical polyprenyl diphosphate synthases and were 528, 440, and 537 amino acids in length in B. albus, S. complicata, and R. minuta, respectively. Escherichia coli expressing the fungal dps1 genes produced CoQ10 in addition to endogenous CoQ8. Two of the three fungal dps1 genes (from S. complicata and R. minuta) were able to replace the function of ispB in an E. coli mutant strain. In vitro enzymatic activities were also detected in recombinant strains. The three dps1 genes were able to complement a Schizosaccharomyces pombe dps1, dlp1 double mutant. Recombinant S. pombe produced mainly CoQ10, indicating that the introduced genes were independently functional and did not require dlp1. The cloning of dps1 genes from various fungi has the potential to enhance production of CoQ10 in other organisms.
Assuntos
Alquil e Aril Transferases/metabolismo , Fungos/enzimologia , Alquil e Aril Transferases/genética , Schizosaccharomyces/enzimologia , Ubiquinona/metabolismoRESUMO
The cAMP-dependent protein kinase Pka1 is known as a regulator of glycogenesis, meiosis, and stress responses in Schizosaccharomyces pombe. We demonstrated that Pka1 is responsible for calcium tolerance. Loss of functional components of the PKA pathway such as Git3, Gpa2, Cyr1, and Pka1 yields a CaCl2-sensitive phenotype, while loss of Cgs1, a regulatory subunit of PKA, results in CaCl2 tolerance. Cytoplasmic distribution of Cgs1 and Pka1 is increased by the addition of CaCl2, suggesting that CaCl2 induces dissociation of Cgs1 and Pka1. The expression of Prz1, a transcriptional regulator in calcium homeostasis, is elevated in a pka1∆ strain and in a wild type strain under glucose-limited conditions. Accordingly, higher expression of Prz1 in the wild type strain results in a CaCl2-sensitive phenotype. These findings suggest that Pka1 is essential for tolerance to exogenous CaCl2, probably because the expression level of Prz1 needs to be properly regulated by Pka1.
Assuntos
Cloreto de Cálcio/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/metabolismo , Fatores de Transcrição/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/deficiência , Proteínas Quinases Dependentes de AMP Cíclico/genética , Ativação Enzimática/efeitos dos fármacos , Deleção de Genes , Homeostase/efeitos dos fármacos , Fenótipo , Transporte Proteico/efeitos dos fármacos , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genéticaRESUMO
Fungal cell walls are medically important since they represent a drug target site for antifungal medication. So far there is no method to directly visualize structurally similar cell wall components such as α-glucan, ß-glucan and mannan with high specificity, especially in a label-free manner. In this study, we have developed a Raman spectroscopy based molecular imaging method and combined multivariate curve resolution analysis to enable detection and visualization of multiple polysaccharide components simultaneously at the single cell level. Our results show that vegetative cell and ascus walls are made up of both α- and ß-glucans while spore wall is exclusively made of α-glucan. Co-localization studies reveal the absence of mannans in ascus wall but are distributed primarily in spores. Such detailed picture is believed to further enhance our understanding of the dynamic spore wall architecture, eventually leading to advancements in drug discovery and development in the near future.
Assuntos
Parede Celular/química , Saccharomyces cerevisiae/fisiologia , Esporos Fúngicos/química , Glucanos/análise , Mananas/análise , Análise Multivariada , Microscopia Óptica não Linear , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismoRESUMO
Coenzyme Q (CoQ) is a component of the electron transport chain that participates in aerobic cellular respiration to produce ATP. In addition, CoQ acts as an electron acceptor in several enzymatic reactions involving oxidation-reduction. Biosynthesis of CoQ has been investigated mainly in Escherichia coli and Saccharomyces cerevisiae, and the findings have been extended to various higher organisms, including plants and humans. Analyses in yeast have contributed greatly to current understanding of human diseases related to CoQ biosynthesis. To date, human genetic disorders related to mutations in eight COQ biosynthetic genes have been reported. In addition, the crystal structures of a number of proteins involved in CoQ synthesis have been solved, including those of IspB, UbiA, UbiD, UbiX, UbiI, Alr8543 (Coq4 homolog), Coq5, ADCK3, and COQ9. Over the last decade, knowledge of CoQ biosynthesis has accumulated, and striking advances in related human genetic disorders and the crystal structure of proteins required for CoQ synthesis have been made. This review focuses on the biosynthesis of CoQ in eukaryotes, with some comparisons to the process in prokaryotes.
Assuntos
Trifosfato de Adenosina/biossíntese , Ataxia Cerebelar/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Encefalomiopatias Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Ubiquinona/biossíntese , Ataxia Cerebelar/genética , Ataxia Cerebelar/patologia , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/patologia , Proteínas Mitocondriais/genética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Oxirredução , Plantas/genética , Plantas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ubiquinona/deficiência , Ubiquinona/genéticaRESUMO
Polypeptone is widely excluded from Schizosaccharomyces pombe growth medium. However, the reasons why polypeptone should be avoided have not been documented. Polypeptone dramatically induced cell lysis in the ura4 deletion mutant when cells approached the stationary growth phase, and this phenotype was suppressed by supplementation of uracil. To determine the specificity of this cell lysis phenotype, we created deletion mutants of other genes involved in de novo biosynthesis of uridine monophosphate (ura1, ura2, ura3, and ura5). Cell lysis was not observed in these gene deletion mutants. In addition, concomitant disruption of ura1, ura2, ura3, or ura5 in the ura4 deletion mutant suppressed cell lysis, indicating that cell lysis induced by polypeptone is specific to the ura4 deletion mutant. Furthermore, cell lysis was also suppressed when the gene involved in coenzyme Q biosynthesis was deleted. This is likely because Ura3 requires coenzyme Q for its activity. The ura4 deletion mutant was sensitive to zymolyase, which mainly degrades (1,3)-beta-D glucan, when grown in the presence of polypeptone, and cell lysis was suppressed by the osmotic stabiliser, sorbitol. Finally, the induction of cell lysis in the ura4 deletion mutant was due to the accumulation of orotidine-5-monophosphate. Cell wall integrity was dramatically impaired in the ura4 deletion mutant when grown in the presence of polypeptone. Because ura4 is widely used as a selection marker in S. pombe, caution needs to be taken when evaluating phenotypes of ura4 mutants.
Assuntos
Deleção de Genes , Peptonas/farmacologia , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Fosfatase Alcalina/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Cromatografia Líquida , Marcadores Genéticos , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Proteínas Luminescentes/metabolismo , Espectrometria de Massas , Modelos Genéticos , Osmose , Fenótipo , Saccharomyces cerevisiaeRESUMO
The cAMP-PKA is the major glucose-sensing pathway that controls sexual differentiation in Schizosaccharomyces pombe. Sequencing from the pka1 locus of recessive sam mutants, in which cells are highly inclined to sexual differentiation, led to the identification of mutations in the pka1 locus in sam5 (pka1-G441E) and sam7 (pka1-G441R). Rst2 and Ste11 proteins were induced and localized to the nucleus of sam5 and sam7 mutants even under rich glucose conditions, indicating that the function of Pka1 was completely abolished by mutations. Pka1-G441E and Pka1-G441R mutant proteins reside in the cytoplasm, even under glucose-rich conditions, while wild-type Pka1 resides in the nucleus, indicating that the functionality of Pka1 is important for its nuclear localization. This is supported by the observation that the Pka1-T356A mutant, which partially lacks Pka1 function, was localized to both the cytoplasm and the nucleus, but an active phosphomimetic Pka1-T356D mutant prtotein was localized to the nucleus under glucose-rich conditions. In addition to the basal phosphorylation of Pka1 at T356, hyperphosphorylation of Pka1 was observed under glucose-starved conditions, and such hyperphosphorylation was not observed in pka1-G441E, pka1-G441R, pka1-T356A or pka1-T356D mutants. As these mutant proteins failed to interact with a regulatory subunit Cgs1, hyperphosphorylation of Pka1 mutant proteins was considered to be dependent on Cgs1 interaction. Consistent with a role for Cgs1 in Pka1 phosphorylation, we detected the formation of a Cgs1-Pka1 complex prior to Pka1 hyperphosphorylation. Together, these results indicate that nuclear localization of Pka1 depends on its activity and hyperphosphorylation of Pka1 depends on Cgs1 interaction.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Citoplasma/metabolismo , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Mutação de Sentido Incorreto , Fosforilação , Mutação Puntual , Subunidades Proteicas , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Nine sam mutants that undergo sexual differentiation without requiring starvation in Schizosaccharomyces pombe were previously isolated. In this study, we identified a nonsense mutation on the pka1 locus in the sam6 mutant. pka1 encodes a catalytic subunit of protein kinase A (PKA). Replacement and overexpression of pka1 suppressed the KCl sensitivity and hyper-mating phenotype of sam6, confirming that sam6 is an allele of pka1. To characterize further the regulation of Pka1, we tested the physical interaction between Pka1 and Cgs1 (a regulatory subunit of PKA). Pka1 and Cgs1 physically interacted under glucose-limited conditions but not under glucose-rich conditions. In addition, the formation of a Pka1-Cgs1 complex was detected under glucose-limited conditions by Blue Native PAGE. Furthermore, the Pka1 protein was found to be phosphorylated under glucose-starved conditions, and at the same time its localization shifted from the nucleus towards the cytoplasm (mainly the vacuoles), suggesting a strong relationship among phosphorylation, complex formation, and the cytoplasmic distribution of Pka1.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Glucose/deficiência , Subunidades Proteicas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Transdução de Sinais/genética , Sequência de Bases , Códon sem Sentido , Proteínas Quinases Dependentes de AMP Cíclico/genética , Citoplasma/genética , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Estudos de Associação Genética , Genótipo , Imunoprecipitação , Dados de Sequência Molecular , Fenótipo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Plasmídeos , Cloreto de Potássio/metabolismo , Cloreto de Potássio/farmacologia , Ligação Proteica , Subunidades Proteicas/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Transdução GenéticaRESUMO
The length of the isoprenoid-side chain in ubiquinone, an essential component of the electron transport chain, is defined by poly-prenyl diphosphate synthase, which comprises either homomers (e.g., IspB in Escherichia coli) or heteromers (e.g., decaprenyl diphosphate synthase (Dps1) and D-less polyprenyl diphosphate synthase (Dlp1) in Schizosaccharomyces pombe and in humans). We found that expression of either dlp1 or dps1 recovered the thermo-sensitive growth of an E. coli ispB(R321A) mutant and restored IspB activity and production of Coenzyme Q-8. IspB interacted with Dlp1 (or Dps1), forming a high-molecular weight complex that stabilized IspB, leading to full functionality.
Assuntos
Alquil e Aril Transferases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Animais , Divisão Celular , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Teste de Complementação Genética , Temperatura Alta , Humanos , Immunoblotting , Camundongos , Peso Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Mutação , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquinona/metabolismoRESUMO
We have constructed coenzyme Q deficient fission yeast strains by deletion of ten different genes, all of which are absolutely required for the CoQ10 biosynthesis. We found that sulfide was highly accumulated in all fission yeast CoQ10 deficient mutants. In fission yeast sulfide is required for the synthesis of cysteine and homocysteine which are catalyzed by cysteine synthase (Cys1a) and homocysteine synthase (Met17), respectively. To better understand the relation between sulfide metabolism and coenzyme Q, we expressed cys1a, met17 and hmt2, which encodes sulfide-quinone oxidoreductase, in CoQ10 deficient mutants and other mutants, and measured the level of sulfide. Although expression of cys1a and met17 lowered sulfide production in CoQ10 deficient mutants, hmt2 did not lower the level of sulfide, because Hmt2 requires coenzyme Q for its function. In contrast, expression of hmt2 lowered sulfide production in cys1a and met17 mutants. These and other results indicate that coenzyme Q is important for sulfide oxidation through sulfide-quinone oxidoreductase to detoxify excess sulfide in fission yeast.
Assuntos
Schizosaccharomyces/metabolismo , Sulfetos/metabolismo , Ubiquinona/deficiência , Cisteína/farmacologia , Sulfeto de Hidrogênio/metabolismo , Quinona Redutases/genética , Quinona Redutases/metabolismo , Schizosaccharomyces/genéticaRESUMO
Homozygous mice carrying kd (kidney disease) mutations in the gene encoding prenyl diphosphate synthase subunit 2 (Pdss2kd/kd) develop interstitial nephritis and eventually die from end-stage renal disease. The PDSS2 polypeptide in concert with PDSS1 synthesizes the polyisoprenyl tail of coenzyme Q (Q or ubiquinone), a lipid quinone required for mitochondrial respiratory electron transport. We have shown that a deficiency in Q content is evident in Pdss2kd/kd mouse kidney lipid extracts by 40 days of age and thus precedes the onset of proteinuria and kidney disease by several weeks. The presence of the kd (V117M) mutation in PDSS2 does not prevent its association with PDSS1. However, heterologous expression of the kd mutant form of PDSS2 together with PDSS1 in Escherichia coli recapitulates the Q deficiency observed in the Pdss2kd/kd mouse. Dietary supplementation with Q10 provides a dramatic rescue of both proteinuria and interstitial nephritis in the Pdss2kd/kd mutant mice. The results presented suggest that Q may be acting as a potent lipid-soluble antioxidant, rather than by boosting kidney mitochondrial respiration. Such Q10 supplementation may have profound and beneficial effects in treatment of certain forms of focal segmental glomerulosclerosis that mirror the renal disease of the Pdss2kd/kd mouse.