RESUMO
Loss of significant intestinal length from congenital anomaly or disease may lead to short bowel syndrome (SBS); intestinal failure may be partially offset by a gain in epithelial surface area, termed adaptation. Current in vivo models of SBS are costly and technically challenging. Operative times and survival rates have slowed extension to transgenic models. We created a new reproducible in vivo model of SBS in zebrafish, a tractable vertebrate model, to facilitate investigation of the mechanisms of intestinal adaptation. Proximal intestinal diversion at segment 1 (S1, equivalent to jejunum) was performed in adult male zebrafish. SBS fish emptied distal intestinal contents via stoma as in the human disease. After 2 wk, S1 was dilated compared with controls and villus ridges had increased complexity, contributing to greater villus epithelial perimeter. The number of intervillus pockets, the intestinal stem cell zone of the zebrafish increased and contained a higher number of bromodeoxyuridine (BrdU)-labeled cells after 2 wk of SBS. Egf receptor and a subset of its ligands, also drivers of adaptation, were upregulated in SBS fish. Igf has been reported as a driver of intestinal adaptation in other animal models, and SBS fish exposed to a pharmacological inhibitor of the Igf receptor failed to demonstrate signs of intestinal adaptation, such as increased inner epithelial perimeter and BrdU incorporation. We describe a technically feasible model of human SBS in the zebrafish, a faster and less expensive tool to investigate intestinal stem cell plasticity as well as the mechanisms that drive intestinal adaptation.
Assuntos
Adaptação Biológica/fisiologia , Intestinos , Síndrome do Intestino Curto/metabolismo , Animais , Antimetabólitos/farmacologia , Bromodesoxiuridina/farmacologia , Proliferação de Células , Procedimentos Cirúrgicos do Sistema Digestório/métodos , Modelos Animais de Doenças , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Mucosa Intestinal/patologia , Intestinos/patologia , Intestinos/fisiopatologia , Intestinos/cirurgia , Masculino , Células-Tronco/fisiologia , Redução de Peso , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Wsc1 and Mid2 are highly O-glycosylated cell surface proteins that reside in the plasma membrane of Saccharomyces cerevisiae. They have been proposed to function as mechanosensors of cell wall stress induced by wall remodeling during vegetative growth and pheromone-induced morphogenesis. These proteins are required for activation of the cell wall integrity signaling pathway that consists of the small G-protein Rho1, protein kinase C (Pkc1), and a mitogen-activated protein kinase cascade. We show here by two-hybrid experiments that the C-terminal cytoplasmic domains of Wsc1 and Mid2 interact with Rom2, a guanine nucleotide exchange factor (GEF) for Rho1. At least with regard to Wsc1, this interaction is mediated by the Rom2 N-terminal domain. This domain is distinct from the Rho1-interacting domain, suggesting that the GEF can interact simultaneously with a sensor and with Rho1. We also demonstrate that extracts from wsc1 and mid2 mutants are deficient in the ability to catalyze GTP loading of Rho1 in vitro, providing evidence that the function of the sensor-Rom2 interaction is to stimulate nucleotide exchange toward this G-protein. In a related line of investigation, we identified the PMT2 gene in a genetic screen for mutations that confer an additive cell lysis defect with a wsc1 null allele. Pmt2 is a member of a six-protein family in yeast that catalyzes the first step in O mannosylation of target proteins. We demonstrate that Mid2 is not mannosylated in a pmt2 mutant and that this modification is important for signaling by Mid2.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Parede Celular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/genética , Ativação Enzimática/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular , Manosiltransferases/química , Manosiltransferases/genética , Manosiltransferases/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Mutação , Feromônios/farmacologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/enzimologia , Transdução de Sinais , Especificidade por Substrato , Técnicas do Sistema de Duplo-HíbridoRESUMO
Xerostomia results from salivary gland irradiation during treatment of head and neck malignancies. In addition to having difficulty with speech and swallowing, these patients experience loss of taste, dental caries, and chronic fungal infections. The paired submandibular glands provide 70 percent of the normal salivary flow and are difficult to shield during radiation therapy. Another sicca condition, xerophthalmia, may result from facial nerve injury or other medical disorders and results in pain, corneal ulceration, and possible vision loss. Treatment options for xerostomia are limited, and management of xerophthalmia usually focuses on the eyelids, rather than the fundamental problem of inadequate secretory protection. In this study, a rat model for submandibular gland microvascular transplantation was developed to assess the feasibility of salivary tissue transfer. Sixteen rats underwent submandibular gland transplantation from the neck to the groin. Fourteen of these rats underwent microvascular anastomosis of the vascular pedicle. Ten glands were assessed for viability at 4 days after transplantation, and four glands were examined after 7, 10, 14, or 21 days. By gross and histologic examination, 93 percent of transplanted glands showed expected long-term viability after at least 4 postoperative days. Microvascular techniques were shown to be applicable to the transplantation of submandibular gland salivary tissue. This has not previously been shown in a rat model. It is possible that submandibular glands could be transplanted to the eye for treatment of xerophthalmia and out of the neck during irradiation of the head and neck, with subsequent replantation after treatment as a means of preventing permanent xerostomia.
Assuntos
Glândula Submandibular/transplante , Anastomose Cirúrgica , Animais , Sobrevivência Celular , Estudos de Viabilidade , Masculino , Microcirurgia , Modelos Animais , Ratos , Glândula Submandibular/patologia , Xerostomia/prevenção & controleRESUMO
The cell integrity pathway of Saccharomyces cerevisiae monitors cell wall remodelling during growth and differentiation. Additionally, this pathway responds to environmental stresses that challenge the integrity of the cell wall. We conducted a genome-wide survey of genes whose expression was altered in response to activation of Mpk1/Slt2, the MAP kinase, under the control of cell integrity signalling. We identified 25 genes whose regulation was altered by Mpk1 activity. Among these, 20 were positively regulated by Mpk1, and five were negatively regulated. Most of the genes identified encode either known or suspected cell wall proteins or enzymes involved in cell wall biogenesis. These include glycosyl-phosphatidylinositol (GPI) proteins, members of the Pir family of cell wall proteins, Mpk1 itself and others. All of the regulation detected was mediated by the Rlm1 transcription factor, a MADS-box protein that is phosphorylated and activated by Mpk1, but for which no transcriptional targets had been identified. A similar pattern of regulation was observed when cell integrity signalling was induced by environmental stress (i.e. temperature upshift).
Assuntos
Parede Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transdução de Sinais , Ativação Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosilfosfatidilinositóis , Proteínas de Domínio MADS , Hibridização de Ácido Nucleico/métodos , RNA Fúngico/genética , RNA Fúngico/isolamento & purificação , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Temperatura , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Hcs77 is a putative cell surface sensor for cell integrity signaling in Saccharomyces cerevisiae. Its loss of function results in cell lysis during growth at elevated temperatures (e.g., 39 degrees C) and impaired signaling to the Mpk1 mitogen-activated protein kinase in response to mild heat shock. We isolated the MID2 gene as a dosage suppressor of the cell lysis defect of an hcs77 null mutant. MID2 encodes a putative membrane protein whose function is required for survival of pheromone treatment. Mid2 possesses properties similar to those of Hcs77, including a single transmembrane domain and a long region that is rich in seryl and threonyl residues. We demonstrate that Mid2 is required for cell integrity signaling in response to pheromone. Additionally, we show that Mid2 and Hcs77 serve a redundant but essential function as cell surface sensors for cell integrity signaling during vegetative growth. Both proteins are uniformly distributed through the plasma membrane and are highly O-mannosylated on their extracellular domains. Finally, we identified a yeast homolog of MID2, designated MTL1, which provides a partially redundant function with MID2 for cell integrity signaling during vegetative growth at elevated temperature but not for survival of pheromone treatment. We conclude that Hcs77 is dedicated to signaling cell wall stress during vegetative growth and that Mid2 participates in this signaling, but its primary role is in signaling wall stress during pheromone-induced morphogenesis.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Membrana/genética , Receptores de Superfície Celular/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transdução de Sinais , Sequência de Aminoácidos , Sobrevivência Celular , Parede Celular/metabolismo , Deleção de Genes , Glicosilação , Proteínas de Fluorescência Verde , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes/metabolismo , Manosidases/farmacologia , Glicoproteínas de Membrana , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutagênese , Feromônios/farmacologia , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , Temperatura , Fatores de Tempo , alfa-ManosidaseRESUMO
FKS1 and FKS2 are alternative subunits of the glucan synthase complex, which is responsible for synthesizing 1,3-beta-glucan chains, the major structural polymer of the Saccharomyces cerevisiae cell wall. Expression of FKS1 predominates during growth under optimal conditions. In contrast, FKS2 expression is induced by mating pheromone, high extracellular [Ca2+], growth on poor carbon sources, or in an fks1 mutant. Induction of FKS2 expression in response to pheromone, CaCl2, or loss of FKS1 function requires the Ca2+/calmodulin-dependent protein phosphatase calcineurin. Therefore, a double mutant in calcineurin (CNB1) and FKS1 is inviable due to a deficiency in FKS2 expression. To identify novel regulators of FKS2 expression, we isolated genes whose overexpression obviates the calcineurin requirement for viability of an fks1 mutant. Two components of the cell integrity signaling pathway controlled by the RHO1 G protein (MKK1 and RLM1) were identified through this screen. This signaling pathway is activated during growth at moderately high temperatures. We demonstrate that calcineurin and the cell integrity pathway function in parallel, through separable promoter elements, to induce FKS2 expression during growth at 39 degrees C. Because RHO1 also serves as a regulatory subunit of the glucan synthase, our results define a regulatory circuit through which RHO1 controls both the activity of this enzyme complex and the expression of at least one of its components. We show also that FKS2 induction during growth on poor carbon sources is a response to glucose depletion and is under the control of the SNF1 protein kinase and the MIG1 transcriptional repressor. Finally, we show that FKS2 expression is induced as cells enter stationary phase through a SNF1-, calcineurin-, and cell integrity signaling-independent pathway.
Assuntos
Calcineurina/metabolismo , Proteínas Fúngicas/biossíntese , Regulação Enzimológica da Expressão Gênica , Glucosiltransferases , Proteínas de Membrana/biossíntese , Proteína Quinase C/metabolismo , Proteínas de Saccharomyces cerevisiae , Cálcio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Indução Enzimática , Glucose/metabolismo , Proteínas de Domínio MADS , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/metabolismo , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Temperatura , Fatores de Transcrição/metabolismo , Transcrição Gênica , Dedos de ZincoRESUMO
The protein kinase C of Saccharomyces cerevisiae, Pkc1, regulates a MAP kinase, Mpk1, whose activity is stimulated at the G1-S transition of the cell cycle and by perturbations to the cell surface, e.g. induced by heat shock. The activity of the Pkc1 pathway is partially dependent on Cdc28 activity. Swi4 activates transcription of many genes at the G1-S transition, including CLN1 and CLN2. We find that swi4 mutants are defective specifically in bud emergence. The growth and budding defects of swi4 mutants are suppressed by overexpression of PKC1. This suppression requires CLN1 and CLN2. Inhibition of the Pkc1 pathway exacerbates the growth and bud emergence defects of swi4 mutants. We find that another dose-dependent suppressor of swi4 mutants, the novel gene HCS77, encodes a putative integral membrane protein. Hcs77 may regulate the Pkc1 pathway; hcs77 mutants exhibit phenotypes like those of mpk1 mutants, are partially suppressed by overexpression of PKC1 and are defective in heat shock induction of Mpk1 activity. We propose that the Pkc1 pathway promotes bud emergence and organized surface growth and is activated by Cdc28-Cln1/Cln2 at the G1-S transition and by Hcs77 upon heat shock. Hcs77 may monitor the state of the cell surface.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/genética , Proteína Quinase C/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Ciclo Celular/fisiologia , Ciclinas/metabolismo , Proteínas de Ligação a DNA , Citometria de Fluxo , Proteínas Fúngicas/fisiologia , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Supressão Genética/genética , Temperatura , Fatores de Transcrição/genética , Transformação Genética/genéticaRESUMO
We have investigated the role of the essential Rho1 GTPase in cell integrity signaling in budding yeast. Conditional rho1 mutants display a cell lysis defect that is similar to that of mutants in the cell integrity signaling pathway mediated by protein kinase C (Pkc1), which is suppressed by overexpression of Pkc1.rho1 mutants are also impaired in pathway activation in response to growth at elevated temperature. Pkc1 co-immunoprecipitates with Rho1 in yeast extracts, and recombinant Rho1 associates with Pkc1 in vitro in a GTP-dependent manner. Recombinant Rho1 confers upon Pkc1 the ability to be stimulated by phosphatidylserine, indicating that Rho1 controls signal transmission through Pkc1.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteína Quinase C/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas rho de Ligação ao GTP , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Ativação Enzimática , GTP Fosfo-Hidrolases/isolamento & purificação , Proteínas de Ligação ao GTP/isolamento & purificação , Guanosina Trifosfato/metabolismo , Modelos Biológicos , Mutagênese Sítio-Dirigida , Proteína Quinase C/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae , Temperatura , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTPRESUMO
1,3-beta-D-glucan synthase [also known as beta(1-->3) glucan synthase] is a multi-enzyme complex that catalyzes the synthesis of 1,3-beta-linked glucan, a major structural component of the yeast cell wall. Temperature-sensitive mutants in the essential Rho-type guanosine triphosphatase (GTPase), Rho1p, displayed thermolabile glucan synthase activity, which was restored by the addition of recombinant Rho1p. Glucan synthase from mutants expressing constitutively active Rho1p did not require exogenous guanosine triphosphate for activity. Rho1p copurified with beta(1-->3)glucan synthase and associated with the Fks1p subunit of this complex in vivo. Both proteins were localized predominantly at sites of cell wall remodeling. Therefore, it appears that Rho1p is a regulatory subunit of beta(1-->3)glucan synthase.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Glucosiltransferases/metabolismo , Proteínas de Membrana , Complexos Multienzimáticos/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Schizosaccharomyces pombe , Proteínas rho de Ligação ao GTP , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/isolamento & purificação , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/isolamento & purificação , Glucosiltransferases/química , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Trifosfato/metabolismo , Complexos Multienzimáticos/química , Proteína Quinase C/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae , TemperaturaRESUMO
In the budding yeast, Saccharomyces cerevisiae, four separate but structurally related mitogen-activated protein kinase (MAPK) activation pathways are known. The best understood of these regulates mating. Pheromone binding to receptor informs cells of the proximity of a mating partner and induces differentiation to a mating competent state. The MAPK activation cascade mediating this signal is made up of Ste11 (a MEK kinase [MEKK]), Ste7 (a MAPK/ERK kinase [MEK]), and the redundant MAPK-related Fus3 and Kss1 enzymes. Another MAPK activation pathway is important for cell integrity and regulates cell wall construction. This cascade consists of Bck1 (a MEKK), the redundant Mkk1 and Mkk2 enzymes (MEKs), and Mpk1 (a MAPK). We exploited these two pathways to learn about the coordination and signal transmission fidelity of MAPK activation cascades. Two lines of evidence suggest that the activities of the mating and cell integrity pathways are coordinated during mating differentiation. First, cells deficient in Mpk1 are susceptible to lysis when they make a mating projection in response to pheromone. Second, Mpk1 activation during pheromone induction coincides with projection formation. The mechanism underlying this coordination is still unknown to us. Our working model is that projection formation generates a mobile second messenger for activation of the cell integrity pathway. Analysis of a STE7 mutation gave us some unanticipated but important insights into parameters important for fidelity of signal transmission. The Ste7 variant has a serine to proline substitution at position 368. Ste7-P368 has higher basal activity than the wild-type enzyme but still requires Ste11 for its function. Additionally, the proline substitution enables the variant to transmit the signal from mammalian Raf expressed in yeast. This novel activity suggests that Ste7-P368 is inherently more permissive than Ste7 in its interactions with MEKKs. Yet, Ste7-P368 cross function in the cell integrity pathway occurs only when it is highly overproduced or when Ste5 is missing. This behavior suggests that Ste5, which has been proposed to be a tether for the kinases in the mating pathway, contributes to Ste7 specificity and fidelity of signal transmission.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Saccharomyces cerevisiae/enzimologia , Transdução de Sinais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , MutaçãoRESUMO
Yeast cells respond to hypertonic shock by activation of a (MAP) mitogen-activated protein kinase cascade called the (HOG) high osmolarity glycerol response pathway. How yeast respond to hypotonic shock is unknown. Results of this investigation show that a second MAP kinase cascade in yeast called the protein kinase C1 (PKC1) pathway is activated by hypotonic shock. Tyrosine phosphorylation of the PKC1 pathway MAP kinase increased rapidly in cells following a shift of the external medium to lower osmolarity. The intensity of the response was proportional to the magnitude of the decrease in extracellular osmolarity. This response to hypotonic shock required upstream protein kinases of the PKC1 pathway. Increasing external osmolarity inhibited tyrosine phosphorylation of the PKC1 pathway MAP kinase, a response that was blocked by BCK1-20, a constitutively active mutant in an upstream protein kinase. These results indicate that yeast contain two osmosensing signal transduction pathways, the HOG pathway and the PKC1 pathway, that respond to hypertonic and hypotonic shock, respectively.
Assuntos
Proteína Quinase C/metabolismo , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ativação Enzimática , Glucose/farmacologia , Soluções Hipertônicas , Concentração Osmolar , Fosforilação , Fosfotirosina/análise , Saccharomyces cerevisiae/enzimologia , Solução Salina HipertônicaRESUMO
The PKC1 gene of budding yeast encodes a homolog of the alpha, beta, and gamma isoforms of mammalian PKC that is proposed to regulate a MAPK-activation pathway. Mutants in this pathway undergo cell lysis resulting from a deficiency in cell wall construction when they attempt to grow at elevated temperatures. We show that the PKC1-regulated pathway is important for induced thermotolerance and that the MPK1 protein kinase (the MAPK of this pathway) is strongly activated by mild heat shock. This activation is sustained during growth at high temperature and is dependent on the function of pathway components proposed to function upstream of MPK1, including PKC1. Expression of genes under the control of known heat shock-inducible promoter elements (HSEs and STREs) was not compromised in PKC1 pathway mutants, indicating that this pathway mediates a novel aspect of the yeast heat shock response. We propose that the heat-induced signal for pathway activation is generated in response to weakness in the cell wall created during growth under thermal stress, perhaps as a result of increased membrane fluidity. Evidence is presented that the mechanism by which the cell detects this weakness is by measuring stretch of the plasma membrane.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/biossíntese , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Proteína Quinase C/biossíntese , Saccharomyces cerevisiae/enzimologia , Sequência de Bases , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Catalase/biossíntese , Catalase/metabolismo , Membrana Celular/metabolismo , Primers do DNA , Ativação Enzimática , Genes Fúngicos , Genótipo , Temperatura Alta , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Saccharomyces cerevisiae/genética , Transdução de Sinais , beta-Galactosidase/biossíntese , beta-Galactosidase/metabolismoRESUMO
Mitogen-activated protein kinases function in at least five, physiologically distinct, signaling pathways in yeast. These include pathways that mediate response to mating pheromone, pseudohyphal development and invasive growth, cell integrity, sporulation, and response to high extracellular osmolarity. These kinases and their upstream activating kinases comprise signaling modules that, in at least some cases, exist as multiprotein complexes. Studies during the past year have revealed that the Ste5 protein of the mating pheromone response pathway serves as a scaffold to promote interactions among the protein kinases in this pathway, and to prevent their interaction with kinases of other modules.
Assuntos
Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/enzimologia , Transdução de Sinais/fisiologia , Ativação Enzimática , Glicerol/metabolismo , Mitógenos , Concentração Osmolar , Proteína Quinase C/fisiologiaRESUMO
Saccharomyces cerevisiae has been used as a model for studying the regulation of protein N-myristoylation. MyristoylCoA:protein N-myristoyl-transferase (Nmt1p), is essential for vegetative growth and uses myristoylCoA as its substrate. MyristoylCoA is produced by the fatty acid synthetase (Fas) complex and by cellular acylCoA synthetases. We have recently isolated three unlinked Fatty Acid Activation (FAA) genes encoding long chain acylCoA synthetases and have now recovered a fourth by genetic complementation. When Fas is active and NMT1 cells are grown on media containing a fermentable carbon source, none of the FAA genes is required for vegetative growth. When Fas is inactivated by a specific inhibitor (cerulenin), NMT1 cells are not viable unless the media is supplemented with long chain fatty acids. Supplementation of cellular myristoylCoA pools through activation of imported myristate (C14:0) is predominantly a function of Faa1p, although Faa4p contributes to this process. Cells with nmt181p need larger pools of myristoylCoA because of the mutant enzyme's reduced affinity for this substrate. Faa1p and Faa4p are required for maintaining the viability of nmt1-181 strains even when Fas is active. Overexpression of Faa2p can rescue nmt1-181 cells due to activation of an endogenous pool of C14:0. This pool appears to be derived in part from membrane phospholipids since overexpression of Plb1p, a nonessential lysophospholipase/phospholipase B, suppresses the temperature-sensitive growth arrest and C14:0 auxotrophy produced by nmt1-181. None of the four known FAAs is exclusively responsible for targeting imported fatty acids to peroxisomal beta-oxidation pathways. Introduction of a peroxisomal assembly mutation, pas1 delta, into isogenic NMT1 and nmt1-181 strains with wild type FAA alleles revealed that when Fas is inhibited, peroxisomes contribute to myristoylCoA pools used by Nmt1p. When Fas is active, a fraction of cellular myristoylCoA is targeted to peroxisomes. A NMT1 strain with deletions of all four FAAs is still viable at 30 degrees C on media containing myristate, palmitate, or oleate as the sole carbon source--indicating that S. cerevisiae contains at least one other FAA which directs fatty acids to beta-oxidation pathways.
Assuntos
Ácidos Graxos/metabolismo , Proteínas Fúngicas/genética , Genes Fúngicos , Ácidos Mirísticos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acil Coenzima A/metabolismo , Sequência de Aminoácidos , Animais , Bactérias/genética , Sequência de Bases , Ácido Graxo Sintases/metabolismo , Proteínas Fúngicas/metabolismo , Humanos , Metabolismo dos Lipídeos , Mamíferos , Dados de Sequência Molecular , Ácido Mirístico , Oligodesoxirribonucleotídeos , Filogenia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: The existence of a Saccharomyces cerevisiae gene encoding a novel isoform of protein kinase C was reported recently in this journal. RESULTS: We demonstrate here that, firstly, the purported PKC2 gene does not reside at the chromosomal location to which it was assigned; secondly, it does not exist as a contiguous sequence in the S. cerevisiae genome; thirdly, some of its reported sequences do exist within other yeast genes; and fourthly, some of its reported sequences, encoding regions of the predicted protein related to protein kinase C, do not exist in any context in the yeast genome. CONCLUSIONS: We conclude from these studies that the PKC2 gene is a composite construction of unrelated yeast and non-yeast sequences.
Assuntos
Genes Fúngicos , Proteína Quinase C/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Fúngico/genética , Proteínas Fúngicas/genética , Genoma Fúngico , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas de Ligação a RNA/genética , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
The Saccharomyces cerevisiae dbr1 mutation has been mapped on the left arm of chromosome XI. XIL is a chromosome arm that was until now rather sparsely populated with accurately mapped markers. On the basis of physical data, the overall order of markers is inverted relative to the existing genetic map of XI. We present tetrad analyses using a variety of markers on XI that indicate that the existing genetic map of XIL should be inverted, at least for the strains in which our mapping was carried out, and probably for other S. cerevisiae strains.
Assuntos
Cromossomos Fúngicos , Genes Fúngicos , Saccharomyces cerevisiae/genética , Mapeamento Cromossômico , Cruzamentos Genéticos , Ligação Genética , Marcadores Genéticos , Genótipo , Mapeamento por RestriçãoRESUMO
Several enzymes with lysophospholipase/phospholipase B activity have been described from the budding yeast Saccharomyces cerevisiae. In vitro, these enzymes are capable of hydrolyzing all phospholipids that can be extracted from yeast cells. Two forms of the enzyme have been isolated from plasma membranes and a third from culture supernatants and the periplasmic space, but their biological roles have not been determined. These highly glycosylated enzymes were reported to have very similar catalytic properties but differed with respect to apparent molecular weight. We isolated a gene from S. cerevisiae, encoding a protein predicted to share 45% amino acid sequence identity with phospholipase B from Penicillium notatum. This yeast gene, designated PLB1, was mapped to the left arm of chromosome VIII. No residual lysophospholipase/phospholipase B activity was detected upon assay of extracts or culture supernatants of a plb1 delta mutant. Thus, either the PLB1 gene encodes all of the previously detected isoforms of phospholipase B or its gene product is required for their expression or activation. Deletion of PLB1 did not result in any apparent phenotypic defect, suggesting either that we failed to identify the growth conditions that would betray such a defect or that Plb1p is functionally redundant with another protein, whose activity has gone undetected. A plb1 delta mutant released wild-type levels of the soluble phosphatidylinositol metabolite glycerophosphoinositol into the growth medium but released greatly reduced levels of the corresponding phosphatidylcholine and phosphatidylethanolamine metabolites. These results indicate that PLB1 is principally responsible for the production of the deacylation products of phosphatidylcholine and phosphatidylethanolamine but not phosphatidylinositol.
Assuntos
Proteínas Fúngicas/genética , Lisofosfolipase/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , DNA Fúngico , Ativação Enzimática , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Proteínas de Membrana , Dados de Sequência Molecular , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de AminoácidosRESUMO
The PKC1 gene of the budding yeast Saccharomyces cerevisiae encodes a homolog of the alpha, beta, and gamma isoforms of mammalian protein kinase C (PKC) that is essential for cell growth. Loss of PKC1 function results in a cell lysis defect that is due to a deficiency in cell wall construction. In this study, Pkc1p was modified at its COOH terminus with the influenza virus hemagglutinin epitope and was detected by SDS-polyacrylamide gel electrophoresis as a 145- and 150-kDa doublet when overproduced in yeast cells. Pkc1p displayed intrinsic Ser/Thr protein kinase activity in vitro, possessing a substrate specificity similar to that described for mammalian PKC. Specifically, preferred substrates possess an arginine at position -3 and a basic residue at position +2 relative to the target site. A catalytically inactive missense mutant of Pkc1p failed to complement a pkc1 delta mutant, suggesting that protein kinase activity is required for the biological function of Pkc1p. Both wild-type Pkc1p and the inactive form were isolated as phosphoproteins, indicating that Pkc1p is phosphorylated in vivo by another protein kinase. In vitro protein kinase activity of Pkc1p was not dependent on activating cofactors normally required for stimulation of mammalian PKC. However, mutational incapacitation of the pseudosubstrate site of Pkc1p resulted in constitutive activation of the enzyme, both in vivo and in vitro, suggesting that Pkc1p is normally regulated by a mechanism similar to that of its mammalian counterparts. The apparent molecular mass and substrate specificity of Pkc1p, together with its failure to respond to activating cofactors, suggest that this enzyme is distinct from an enzyme purified previously from budding yeast that has enzymatic properties similar to those of mammalian PKC.
Assuntos
Genes Fúngicos , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Alcaloides/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , Genótipo , Immunoblotting , Cinética , Mamíferos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Saccharomyces cerevisiae/crescimento & desenvolvimento , Estaurosporina , Especificidade por SubstratoRESUMO
The PKC1 gene of the budding yeast Saccharomyces cerevisiae encodes a homolog of the alpha, beta, and gamma isoforms of mammalian PKC that is essential for cell growth. Loss of PKC1 function results in a cell lysis defect that is suppressed by osmotic stabilizing agents, suggesting a defect in cell wall integrity. In this study, we show that Pkc1p-depleted cells develop holes in their cell walls positioned at their bud tips, the site to which growth is focused during polarized cell growth. This result suggests that pkc1 mutants are deficient in the process of cell wall remodeling during growth. In further support of this model, cells bearing a pkc1 delta mutation, allowed to proliferate in the presence of osmotic stabilizing agents, possessed cell walls that were only 60% as thick as wild-type cell walls. This diminution in cell wall material affected both the beta-glucan layer and the mannoprotein layer. We have exploited the cell lysis defect of pkc1 mutants to identify genes that function within the same signalling pathway at points downstream of PKC1. These genes comprise a protein kinase cascade that culminates in the activation of the MAP kinase homolog Mpk1p. The proposed order of protein kinase function, based on genetic experiments, is Pkc1p to Bck1p to Mkk1/2p to Mpk1p. Consistent with the proposed model, Pkc1p selectively phosphorylates Bck1p in vitro and Mpk1p protein kinase activity requires a functional BCK1 gene.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteína Quinase C/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Divisão Celular/genética , Divisão Celular/fisiologia , Parede Celular/ultraestrutura , Genes Fúngicos , Microscopia Eletrônica , Modelos Biológicos , Mutação , Fosforilação , Proteína Quinase C/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for yeast cell growth. Loss of PKC1 function results in cell lysis due to an inability to remodel the cell wall properly during growth. The PKC1 gene has been proposed to regulate a bifurcated pathway, on one branch of which function four putative protein kinases that catalyze a linear cascade of protein phosphorylation culminating in the activation of the mitogen-activated protein kinase homolog, Mpk1p. Here we describe two genes whose overexpression suppress both an mpk1 delta mutation and a pkc1 delta mutation. One of these genes is identical to the previously identified PPZ2 gene. The PPZ2 gene is predicted to encode a type 1-related protein phosphatase and is functionally redundant with a closely related gene, designated PPZ1. Deletion of both PPZ1 and PPZ2 resulted in a temperature-dependent cell lysis defect similar to that observed for bck1 delta, mkk1,2 delta, or mpk1 delta mutants. However, ppz1,2 delta mpk1 delta triple mutants displayed a cell lysis defect at all temperatures. The additivity of the ppz1,2 delta defect with the mpk1 delta defect, combined with the results of genetic epistasis experiments, suggested either that the PPZ1- and PPZ2-encoded protein phosphatases function on a branch of the PKC1-mediated pathway different from that defined by the protein kinases or that they play an auxiliary role in the pathway. The other suppressor gene, designated BCK2 (for bypass of C kinase), is predicted to encode a 92-kDa protein that is rich in serine and threonine residues. Genetic interactions between BCK2 and other pathway components suggested that BCK2 functions on a common pathway branch with PPZ1 and PPZ2.