RESUMO
Ever since George Palade's pioneering studies of zymogen secretion from pancreatic acinar cells, the underlying molecular mechanisms of vesicle-mediated protein transport have captivated cell biologists and biochemists. A watershed meeting on "Phosphoinositides and the Golgi", held at the Fogarty International Center of the National Institutes of Health in Bethesda, Maryland (March 13-14, 2001), provided reinterpretation and striking new insights about the functions of this phospholipid class in intracellular protein trafficking.
Assuntos
Secreções Corporais/metabolismo , Complexo de Golgi/metabolismo , Fosfatidilinositóis/metabolismo , Transporte Proteico/genética , Vesículas Secretórias/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Complexo de Golgi/genética , Complexo de Golgi/ultraestrutura , Humanos , Fosfatidilinositóis/genética , Estrutura Terciária de Proteína/genética , Vesículas Secretórias/ultraestruturaRESUMO
In metazoans, certain calmodulin-related calcium-binding proteins (recoverins, neurocalcins and frequenins) are found at highest levels in excitable cells, but their physiological roles are largely uncharacterized. Here we show that Saccharomyces cerevisiae contains a frequenin homologue, Frq1, and that its target is Pik1, a phosphatidylinositol-4-OH kinase. Frq1 binds to a conserved sequence motif in Pik1 outside Pik1's catalytic domain and stimulates its activity in vitro. N-myristoylated Frq1 may also assist in Pik1 localization.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , 1-Fosfatidilinositol 4-Quinase/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Proteínas de Ligação ao Cálcio/genética , Sequência Conservada , Ativação Enzimática , Proteínas Fúngicas/genética , Genes Fúngicos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
STE6 gene product is required for secretion of the lipopeptide mating pheromone a-factor by Saccharomyces cerevisiae MATa cells. Radiolabeling and immunoprecipitation, either with specific polyclonal antibodies raised against a TrpE-Ste6 fusion protein or with mAbs that recognize c-myc epitopes in fully functional epitope-tagged Ste6 derivatives, demonstrated that Ste6 is a 145-kD phosphoprotein. Subcellular fractionation, various extraction procedures, and immunoblotting showed that Ste6 is an intrinsic plasma membrane-associated protein. The apparent molecular weight of Ste6 was unaffected by tunicamycin treatment, and the radiolabeled protein did not bind to concanavalin A, indicating that Ste6 is not glycosylated and that glycosylation is not required either for its membrane delivery or its function. The amino acid sequence of Ste6 predicts two ATP-binding folds; correspondingly, Ste6 was photoaffinity-labeled specifically with 8-azido-[alpha-32P]ATP. Indirect immunofluorescence revealed that in exponentially growing MATa cells, the majority of Ste6 showed a patchy distribution within the plasma membrane, but a significant fraction was found concentrated in a number of vesicle-like bodies subtending the plasma membrane. In contrast, in MATa cells exposed to the mating pheromone alpha-factor, which markedly induced Ste6 production, the majority of Ste6 was incorporated into the plasma membrane within the growing tip of the elongating cells. The highly localized insertion of this transporter may establish pronounced anisotropy in a-factor secretion from the MATa cell, and thereby may contribute to the establishment of the cell polarity which restricts partner selection and cell fusion during mating to one MAT alpha cell.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Membrana Celular/fisiologia , Polaridade Celular , Proteínas Fúngicas/metabolismo , Glicoproteínas , Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Epitopos , Proteínas Fúngicas/imunologia , Regulação Fúngica da Expressão Gênica , Fator de Acasalamento , Proteínas de Membrana/imunologia , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Fosfoproteínas/fisiologia , Saccharomyces cerevisiae/ultraestruturaRESUMO
The gene (FPR3) encoding a novel type of peptidylpropyl-cis-trans-isomerase (PPIase) was isolated during a search for previously unidentified nuclear proteins in Saccharomyces cerevisiae. PPIases are thought to act in conjunction with protein chaperones because they accelerate the rate of conformational interconversions around proline residues in polypeptides. The FPR3 gene product (Fpr3) is 413 amino acids long. The 111 COOH-terminal residues of Fpr3 share greater than 40% amino acid identity with a particular class of PPIases, termed FK506-binding proteins (FKBPs) because they are the intracellular receptors for two immunosuppressive compounds, rapamycin and FK506. When expressed in and purified from Escherichia coli, both full-length Fpr3 and its isolated COOH-terminal domain exhibit readily detectable PPIase activity. Both fpr3 delta null mutants and cells expressing FPR3 from its own promoter on a multicopy plasmid have no discernible growth phenotype and do not display any alteration in sensitivity to the growth-inhibitory effects of either FK506 or rapamycin. In S. cerevisiae, the gene for a 112-residue cytosolic FKBP (FPR1) and the gene for a 135-residue ER-associated FKBP (FPR2) have been described before. Even fpr1 fpr2 fpr3 triple mutants are viable. However, in cells carrying an fpr1 delta mutation (which confers resistance to rapamycin), overexpression from the GAL1 promoter of the C-terminal domain of Fpr3, but not full-length Fpr3, restored sensitivity to rapamycin. Conversely, overproduction from the GAL1 promoter of full-length Fpr3, but not its COOH-terminal domain, is growth inhibitory in both normal cells and fpr1 delta mutants. In fpr1 delta cells, the toxic effect of Fpr3 overproduction can be reversed by rapamycin. Overproduction of the NH2-terminal domain of Fpr3 is also growth inhibitory in normal cells and fpr1 delta mutants, but this toxicity is not ameliorated in fpr1 delta cells by rapamycin. The NH2-terminal domain of Fpr3 contains long stretches of acidic residues alternating with blocks of basic residues, a structure that resembles sequences found in nucleolar proteins, including S. cerevisiae NSR1 and mammalian nucleolin. Indirect immunofluorescence with polyclonal antibodies raised against either the NH2- or the COOH-terminal segments of Fpr3 expressed in E. coli demonstrated that Fpr3 is located exclusively in the nucleolus.
Assuntos
Isomerases de Aminoácido/metabolismo , Proteínas de Transporte/metabolismo , Nucléolo Celular/enzimologia , Genes Fúngicos , Polienos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tacrolimo/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/genética , Clonagem Molecular , DNA Fúngico/isolamento & purificação , DNA Fúngico/metabolismo , Escherichia coli , Genótipo , Proteínas de Choque Térmico/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Peptidilprolil Isomerase , Polienos/farmacologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Sirolimo , Proteínas de Ligação a TacrolimoRESUMO
The PAL1 gene was isolated using PCR and degenerate oligonucleotide primers corresponding to highly conserved amino acid sequence motifs diagnostic of the ATP-binding cassette domain of the superfamily of membrane-bound transport proteins typified by mammalian multidrug resistance transporter 1 and Saccharomyces cerevisiae Ste6. The deduced PAL1 gene product is similar in length to, has the same predicted topology as, and shares the highest degree of amino acid sequence identity with two human proteins, adrenoleukodystrophy protein and peroxisomal membrane protein (70 kD), which are both presumptive ATP-binding cassette transporters thought to be constituents of the peroxisomal membrane. As judged by hybridization of a PAL1 probe to isolated RNA and by expression of a PAL1-lacZ fusion, a PAL1 transcript was only detectable when cells were grown on oleic acid, a carbon source which requires the biogenesis of functional peroxisomes for its metabolism. A pal1delta mutant grew normally on either glucose- or glycerol-containing media; however, unlike PAL1+ cells (or the pal1delta mutant carrying the PAL1 gene on a plasmid), pal1delta cells were unable to grow on either a solid medium or a liquid medium containing oleic acid as the sole carbon source. Antibodies raised against a chimeric protein in which the COOH-terminal domain of Pal1 was fused to glutathione S-transferase specifically recognized a protein in extracts from wild-type cells only when grown on oleic acid; this species represents the PAL1 gene product because it was missing in pal1delta cells and more abundant in pal1delta cells expressing PAL1 from a multicopy plasmid. The Pal1 polypeptide was highly enriched in the organellar pellet fraction prepared from wild-type cells by differential centrifugation and comigrated upon velocity sedimentation in a Nycodenz gradient with a known component of the peroxisomal matrix, e-oxoacyl-CoA thiolase. As judged by both subcellular fractionation and indirect immunofluorescence, localization of 3-oxoacyl-CoA thiolase to peroxisomes was unchanged whether Pal1 was present, absent, or overexpressed. These findings demonstrate that Pal1 is a peroxisome-specific protein, that it is required for peroxisome function, but that it is not necessary for the biogenesis of peroxisomes or for the import of 3-oxoacyl-CoA thiolase (and at least two other peroxisomal matrix proteins).
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas Fúngicas/genética , Microcorpos/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Humanos , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
Extracts from BSC-40 cells infected with vaccinia recombinants expressing either the yeast KEX2 prohormone endoprotease or a human structural homologue (fur gene product) contained an elevated level of a membrane-associated endoproteolytic activity that could cleave at pairs of basic amino acids (-LysArg- and -ArgArg-). The fur-directed activity (furin) shared many properties with Kex2p including activity at pH 7.3 and a requirement for calcium. By using antifurin antibodies, immunoblot analysis detected two furin translation products (90 and 96 kD), while immunofluorescence indicated localization to the Golgi apparatus. Coexpression of either Kex2p or furin with the mouse beta-nerve growth factor precursor (pro-beta-NGF) resulted in greatly enhanced conversion of the precursor to mature nerve growth factor. Thus, the sequence homology shared by furin and the yeast KEX2 prohormone processing enzyme is reflected by significant functional homology both in vitro and in vivo.
Assuntos
Endopeptidases/genética , Proteínas de Membrana/genética , Fatores de Crescimento Neural/metabolismo , Pró-Proteína Convertases , Precursores de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Subtilisinas/genética , Subtilisinas/fisiologia , Sequência de Aminoácidos , Endopeptidases/fisiologia , Proteínas Fúngicas/genética , Furina , Complexo de Golgi/enzimologia , Humanos , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Saccharomyces cerevisiae/enzimologia , Serina Endopeptidases/fisiologia , Especificidade por SubstratoRESUMO
Saccharomyces cerevisiae was examined for tyrosine kinase activity in vitro because this organism offers molecular and genetic approaches for analyzing the role of tyrosine phosphorylation in cellular growth control that are unavailable in higher eukaryotes. Yeast extracts phosphorylated a random copolymer (glutamic acid:tyrosine, 80:20) at tyrosine in a reaction that was linear with respect to time and protein concentration. In the absence of added copolymer, phosphotyrosine was 0.1 percent of the total phosphoamino acids labeled with [gamma-32P]adenosine triphosphate in endogenous yeast proteins. However, specific activities of these reactions were low (approximately 1 percent of those in extracts of chick embryo fibroblasts). Lack of significant incorporation of label from [alpha-32P]adenosine triphosphate into the copolymer or endogenous yeast proteins demonstrated that nucleotide interconversion, adenylylation, and subsequent hydrolysis could not account for the generation of phosphotyrosine observed.
Assuntos
Proteínas Tirosina Quinases/metabolismo , Saccharomyces cerevisiae/enzimologia , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Peptídeos/metabolismo , Fosforilação , Fosfotirosina , Saccharomyces cerevisiae/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
The prohormone-processing endoprotease (KEX2 gene product) of the yeast Saccharomyces cerevisiae is a membrane-bound, 135,000-dalton glycoprotein, which contains both asparagine-linked and serine- and threonine-linked oligosaccharide and resides in a secretory compartment. Analysis of mutant kex2 genes truncated at their 3' end indicates that carboxyl terminal domains of the enzyme are required for its proper localization within the cell. A human gene product, "furin," shares 50% identity with the catalytic domain of Kex2 protease and is, therefore, a candidate for a human prohormone-processing enzyme.
Assuntos
Pró-Proteína Convertases , Proteínas de Saccharomyces cerevisiae , Serina Endopeptidases/genética , Subtilisinas , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Mutação , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência do Ácido Nucleico , Serina Endopeptidases/metabolismo , Frações Subcelulares/enzimologiaRESUMO
Ste5 is a scaffold for the mitogen-activated protein kinase (MAPK) cascade components in a yeast pheromone response pathway. Ste5 also associates with Ste4, the beta subunit of a heterotrimeric guanine nucleotide-binding protein, potentially linking receptor activation to stimulation of the MAPK cascade. A RING-H2 motif at the Ste5 amino terminus is apparently essential for function because Ste5(C177S) and Ste5(C177A C180A) mutants did not rescue the mating defect of a ste5Delta cell. In vitro Ste5(C177A C180A) bound each component of the MAPK cascade, but not Ste4. Unlike wild-type Ste5, the mutant did not appear to oligomerize; however, when fused to a heterologous dimerization domain (glutathione S-transferase), the chimeric protein restored mating in an ste5Delta cell and an ste4Delta ste5Delta double mutant. Thus, the RING-H2 domain mediates Ste4-Ste5 interaction, which is a prerequisite for Ste5-Ste5 self-association and signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Subunidades beta da Proteína de Ligação ao GTP , Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP , Peptídeos/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Sítios de Ligação , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Dimerização , Proteínas Fúngicas/genética , Teste de Complementação Genética , Glutationa Transferase/química , Fator de Acasalamento , Dados de Sequência Molecular , Feromônios/fisiologia , Mutação Puntual , Polímeros , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Transformação GenéticaRESUMO
alpha-Factor, a tridecapeptide mating pheromone of yeast (Saccharomyces cerevisiae), has extensive sequence homology with the hypothalamic decapeptide gonadotropin-releasing hormone (GnRH). Both synthetic and natural preparations of alpha-mating factor were found to bind specifically to rat pituitary GnRH receptors and to stimulate the release of luteinizing hormone from cultured gonadotrophs. The ability of the yeast pheromone to reproduce the biological actions of GnRH in the mammalian pituitary gland indicates that the structural and functional properties of GnRH-related peptides may have been highly conserved during evolution.
Assuntos
Peptídeos/farmacologia , Receptores de Superfície Celular/efeitos dos fármacos , Saccharomyces cerevisiae/análise , Animais , Relação Dose-Resposta a Droga , Hormônio Luteinizante/metabolismo , Fator de Acasalamento , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Ratos , Receptores LHRHRESUMO
To facilitate functional and mechanistic studies of receptor-G protein interactions, [corrected] the human beta 2-adrenergic receptor (h beta-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h beta-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h beta-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h beta-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by beta-adrenergic receptor agonists was achieved in cells coexpressing h beta-AR and a mammalian G protein (Gs) alpha subunit-demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Receptores Adrenérgicos beta/fisiologia , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais , Sequência de Aminoácidos , Sequência de Bases , Membrana Celular/fisiologia , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Humanos , Iodocianopindolol , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Pindolol/análogos & derivados , Pindolol/metabolismo , Plasmídeos , Regiões Promotoras Genéticas , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae/genéticaRESUMO
The activity of an oncoprotein and the secretion of a pheromone can be affected by an unusual protein modification. Specifically, posttranslational modification of yeast a-factor and Ras protein requires an intermediate of the cholesterol biosynthetic pathway. This modification is apparently essential for biological activity. Studies of yeast mutants blocked in sterol biosynthesis demonstrated that the membrane association and biological activation of the yeast Ras2 protein require mevalonate, a precursor of sterols and other isoprenes such as farnesyl pyrophosphate. Furthermore, drugs that inhibit mevalonate biosynthesis blocked the in vivo action of oncogenic derivatives of human Ras protein in the Xenopus oocyte assay. The same drugs and mutations also prevented the posttranslational processing and secretion of yeast a-factor, a peptide that is farnesylated. Thus, the mevalonate requirement for Ras activation may indicate that attachment of a mevalonate-derived (isoprenoid) moiety to Ras proteins is necessary for membrane association and biological function. These observations establish a connection between the cholesterol biosynthetic pathway and transformation by the ras oncogene and offer a novel pharmacological approach to investigating, and possibly controlling, ras-mediated malignant transformations.
Assuntos
Proteínas Fúngicas/metabolismo , Genes ras , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Supressão Genética , Proteínas ras , Sequência de Aminoácidos , Animais , Células Cultivadas , Drosophila , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/genética , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Sintase/genética , Immunoblotting , Fator de Acasalamento , Ácido Mevalônico/biossíntese , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Testes de Precipitina , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , XenopusRESUMO
Mammalian cell lines (BSC-40, NG108-15, and GH4C1) that cannot process the murine neuroendocrine peptide precursor prepro-opiomelanocortin (mPOMC) when its synthesis is directed by a vaccinia virus vector were coinfected with a second recombinant vaccinia virus carrying the yeast KEX2 gene, which encodes an endopeptidase that cleaves at pairs of basic amino acid residues. mPOMC was cleaved intracellularly to a set of product peptides normally found in vivo, including mature gamma-lipotropin and beta-endorphin1-31. In GH4C1 cells (a rat pituitary line), product peptides were incorporated into stored secretory granules. These results suggest that the inability of any particular cell line to process a prohormone precursor is due to the absence of a suitable endogenous processing enzyme.
Assuntos
Endopeptidases/metabolismo , Pró-Opiomelanocortina/metabolismo , Precursores de Proteínas/metabolismo , Linhagem Celular , Clonagem Molecular , DNA Recombinante , Técnicas In Vitro , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/enzimologiaRESUMO
Phosphatidylinositol (PtdIns) 4-kinase catalyzes the first step in the biosynthesis of PtdIns-4,5-bisphosphate (PtdIns[4,5]P2). Hydrolysis of PtdIns[4,5]P2 in response to extracellular stimuli is thought to initiate intracellular signaling cascades that modulate cell proliferation and differentiation. The PIK1 gene encoding a PtdIns 4-kinase from the yeast Saccharomyces cerevisiae was isolated by polymerase chain reaction (PCR) with oligonucleotides based on the sequence of peptides derived from the purified enzyme. The sequence of the PIK1 gene product bears similarities to that of PtdIns 3-kinases from mammals (p110) and yeast (Vps34p). Expression of PIK1 from a multicopy plasmid elevated PtdIns 4-kinase activity and enhanced the response to mating pheromone. A pik1 null mutant was inviable, indicating that PtdIns4P and presumably PtdIns[4,5]P2 are indispensable phospholipids.
Assuntos
Genes Fúngicos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , 1-Fosfatidilinositol 4-Quinase , Sequência de Aminoácidos , Clonagem Molecular , Expressão Gênica , Dados de Sequência Molecular , Peso Molecular , Mutação , Fosfatidilinositol 4,5-Difosfato , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/química , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
In this review, we will emphasize the role of ATP-dependent membrane transporters in protein export and intracellular protein trafficking in prokaryotic and eukaryotic cells. ATP-binding-cassette (ABC)-transport proteins, also termed "traffic ATPases," belong to a superfamily of ubiquitous ATP-driven membrane transporters that share extensive sequence similarity and highly conserved domain organization. They are implicated in a remarkable variety of transmembrane transport processes, including the transport of ions, heavy metals, sugars, anticancer drugs, amino acids, oligopeptides, and proteins. Bacterial ABC-proteins include the well-characterized periplasmic permeases involved in nutrient uptake, but also include protein secretion systems, such as the exporter for the Escherichia coli enterotoxin hemolysin A. Prominent eukaryotic members of this superfamily include the human P-glycoprotein (which is associated with the phenomenon of multiple drug resistance in tumor cells), the product of the cystic fibrosis gene (CFTR), the gene (pfmdr) implicated in chloroquine resistance of the malarial parasite, putative peptide transporters encoded at the locus for the class II major histocompatibility complex (MHC), and the yeast Ste6 transporter which mediates export of a peptide hormone that lacks a classical hydrophobic signal peptide. The well-established function of prokaryotic ABC-transporters in the secretion of proteins without typical signal sequences, and the example set by the Ste6 transporter, have led to the reasonable hypothesis that certain ABC-proteins in animal cells may be operating by a similar mechanism to mediate the export of a new class of secretory proteins, those lacking a classical hydrophobic signal peptide.
Assuntos
Trifosfato de Adenosina/farmacologia , Proteínas de Transporte/fisiologia , Membrana Celular/metabolismo , Peptídeos/metabolismo , Sinais Direcionadores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Humanos , Fator de Acasalamento , Dados de Sequência MolecularRESUMO
BACKGROUND: In animal cells, recruitment of phosphatidylinositol 3-kinase by growth factor receptors generates 3-phosphoinositides, which stimulate 3-phosphoinositide-dependent protein kinase-1 (PDK1). Activated PDK1 then phosphorylates and activates downstream protein kinases, including protein kinase B (PKB)/c-Akt, p70 S6 kinase, PKC isoforms, and serum- and glucocorticoid-inducible kinase (SGK), thereby eliciting physiological responses. RESULTS: We found that two previously uncharacterised genes of Saccharomyces cerevisiae, which we term PKH1 and PKH2, encode protein kinases with catalytic domains closely resembling those of human and Drosophila PDK1. Both Pkh1 and Pkh2 were essential for cell viability. Expression of human PDK1 in otherwise inviable pkh1Delta pkh2Delta cells permitted growth. In addition, the yeast YPK1 and YKR2 genes were found to encode protein kinases each with a catalytic domain closely resembling that of SGK; both Ypk1 and Ykr2 were also essential for viability. Otherwise inviable ypk1Delta ykr2Delta cells were fully rescued by expression of rat SGK, but not mouse PKB or rat p70 S6 kinase. Purified Pkh1 activated mammalian SGK and PKBalpha in vitro by phosphorylating the same residue as PDK1. Pkh1 activated purified Ypk1 by phosphorylating the equivalent residue (Thr504) and was required for maximal Ypk1 phosphorylation in vivo. Unlike PKB, activation of Ypk1 and SGK by Pkh1 did not require phosphatidylinositol 3,4,5-trisphosphate, consistent with the absence of pleckstrin homology domains in these proteins. The phosphorylation consensus sequence for Ypk1 was similar to that for PKBalpha and SGK. CONCLUSIONS: Pkh1 and Pkh2 function similarly to PDK1, and Ypk1 and Ykr2 to SGK. As in animal cells, these two groups of yeast kinases constitute two tiers of a signalling cascade required for yeast cell growth.
Assuntos
Proteínas Nucleares , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Sequência de Aminoácidos , Animais , Sequência Consenso , Drosophila/enzimologia , Drosophila/genética , Genes Essenciais , Genes Fúngicos , Humanos , Proteínas Imediatamente Precoces , Mamíferos , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Schizosaccharomyces/enzimologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The structural gene for alpha-aminoadipate reductase (LYS2) was isolated from a Saccharomyces cerevisiae genomic DNA library by complementation of a lys2 mutant. Both genetic and biochemical criteria confirmed that the DNA obtained corresponds to the LYS2 locus on chromosome II. Subcloning and deletion analysis showed that a functional LYS2 gene is contained within a 4.6-kilobase (kb) EcoRI-HindIII fragment of the original insert, and the slightly larger EcoRI-ClaI segment (4.8 kb) was used to construct a series of cloning vehicles, including integrating, episomal, replicative, and centromeric vectors. The cloned DNA was also used to generate a genomic deletion that lacks all LYS2 coding sequences on chromosome II. The level of the LYS2 transcript (4.2 kb) was 10-fold higher in cells grown on minimal medium than in cells grown on complete medium and was not repressed by the presence of lysine alone. Gene disruption, gene replacement, and promoter analysis of the major alpha-factor structural gene (MF alpha 1) were performed to illustrate the utility of the LYS2 gene for the genetic manipulation of yeasts. Because all fungi synthesize lysine via the alpha-aminoadipate pathway, the techniques developed here for using the S. cerevisiae LYS2 gene should be directly applicable to other fungal systems.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II , Saccharomyces cerevisiae/genética , Aldeído Oxirredutases/genética , Deleção Cromossômica , Mapeamento Cromossômico , Enzimas de Restrição do DNA/metabolismo , Desoxirribonuclease EcoRI , Desoxirribonuclease HindIII , Genes , L-Aminoadipato-Semialdeído Desidrogenase , Mutação , Plasmídeos , Transcrição GênicaRESUMO
Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphatidylinositol-specific phospholipase C (PI-PLC) generates two second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. The polymerase chain reaction was used to isolate a Saccharomyces cerevisiae gene (PLC1) that encodes a protein of 869 amino acids (designated Plc1p) that bears greatest resemblance to the delta isoforms of mammalian PI-PLC in terms of overall sequence similarity and domain arrangement. Plc1p contains the conserved X and Y domains found in all higher eukaryotic PI-PLCs (51 and 29% identity, respectively, to the corresponding domains of rat delta 1 PI-PLC) and also contains a presumptive Ca(2+)-binding site (an E-F hand motif). Plc1p, modified by in-frame insertion of a His6 tract and a c-myc epitope near its amino terminus, was overexpressed from the GAL1 promoter, partially purified by nickel chelate affinity chromatography, and shown to be an active PLC enzyme in vitro with properties similar to those of its mammalian counterparts. Plc1p activity was strictly Ca2+ dependent: at a high Ca2+ concentration (0.1 mM), the enzyme hydrolyzed PIP2 at a faster rate than phosphatidylinositol, and at a low Ca2+ concentration (0.5 microM), it hydrolyzed PIP2 exclusively. Cells carrying either of two different deletion-insertion mutations (plc1 delta 1::HIS3 and plc1 delta 2::LEU2) were viable but displayed several distinctive phenotypes, including temperature-sensitive growth (inviable above 35 degrees C), osmotic sensitivity, and defects in the utilization of galactose, raffinose, and glycerol at permissive temperatures (23 to 30 degrees C). The findings reported here suggest that hydrolysis of PIP2 in S. cerevisiae is required for a number of nutritional and stress-related responses.
Assuntos
Diester Fosfórico Hidrolases/genética , Saccharomyces cerevisiae/genética , Fosfolipases Tipo C/genética , Sequência de Aminoácidos , Sequência de Bases , Divisão Celular , Clonagem Molecular , Análise Mutacional de DNA , Expressão Gênica , Genes Fúngicos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Diester Fosfórico Hidrolases/metabolismo , RNA Mensageiro/genética , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologia , Alinhamento de Sequência , Deleção de Sequência , Relação Estrutura-Atividade , Fosfolipases Tipo C/metabolismoRESUMO
By using an assay specific for detection of calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase, this enzyme was purified approximately 5,000-fold from extracts of the yeast Saccharomyces cerevisiae. Cna1p and Cna2p, the products of two yeast genes encoding the catalytic (A) subunits of calcineurin, were major constituents of the purified fraction. A third prominent component of apparent molecular mass 16 kDa displayed several properties, including ability to bind 45Ca2+, that are characteristic of the regulatory (B) subunit of mammalian calcineurin and was recognized by an antiserum raised against bovine calcineurin. These antibodies were used to isolate the structural gene (CNB1) encoding this protein from a yeast expression library in the vector lambda gt11. The nucleotide sequence of CNB1 predicted a polypeptide similar in length and highly related in amino acid sequence (56% identity) to the mammalian calcineurin B subunit. Like its counterpart in higher cells, yeast Cnb1p was myristoylated at its N terminus. Mutants lacking Cnb1p, or all three calcineurin subunits (Cna1p, Cna2p, and Cnb1p), were viable. Extracts of cnb1 delta mutants contained no detectable calcineurin activity, even though Cna1p and Cna2p were present at normal levels, suggesting that the B subunit is required for full enzymatic activity in vitro. As was observed previously for MATa cna1 cna2 double mutants, MATa cnb1 mutants were defective in their ability to recover from alpha-factor-induced growth arrest. Thus, the B subunit also is required for the function of calcineurin in promoting adaptation of haploid yeast cells to pheromone in vivo.
Assuntos
Proteínas de Ligação a Calmodulina/genética , Genes Virais , Peptídeos/farmacologia , Feromônios/farmacologia , Fosfoproteínas Fosfatases/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Calcineurina , Proteínas de Ligação a Calmodulina/isolamento & purificação , Proteínas de Ligação a Calmodulina/metabolismo , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Humanos , Soros Imunes , Cinética , Substâncias Macromoleculares , Fator de Acasalamento , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/imunologia , Fosfoproteínas Fosfatases/isolamento & purificação , Fosfoproteínas Fosfatases/metabolismo , Proteínas Recombinantes/farmacologia , Mapeamento por Restrição , Saccharomyces cerevisiae/efeitos dos fármacos , Homologia de Sequência do Ácido NucleicoRESUMO
Exposure of a haploid yeast cell to mating pheromone induces transcription of a set of genes. Induction is mediated through a cis-acting DNA sequence found upstream of all pheromone-responsive genes. Although the STE12 gene product binds specifically to this sequence element and is required for maximum levels of both basal and induced transcription, not all pheromone-responsive genes are regulated in an identical manner. To investigate whether additional factors may play a role in transcription of these genes, a genetic screen was used to identify mutants able to express pheromone-responsive genes constitutively in the absence of Ste12. In this way, we identified a recessive, single gene mutation (mot1, for modifier of transcription) which increases the basal level of expression of several, but not all, pheromone-responsive genes. The mot1-1 allele also relaxes the requirement for at least one other class of upstream activating sequence and enhances the expression of another gene not previously thought to be involved in the mating pathway. Cells carrying mot1-1 grow slowly at 30 degrees C and are inviable at 38 degrees C. The MOT1 gene was cloned by complementation of this temperature-sensitive lethality. Construction of a null allele confirmed that MOT1 is an essential gene. MOT1 residues on chromosome XVI and encodes a large protein of 1,867 amino acids which contains all seven of the conserved domains found in known and putative helicases. The product of MOT1 is strikingly homologous to the Saccharomyces cerevisiae SNF2/SW12 and RAD54 gene products over the entire helicase region.