RESUMO
In Saccharomyces cerevisiae the CDC14 gene is essential for cell cycle progression. Strains carrying the cdc14-1(ts) allele enter the cell cycle and arrest at restrictive temperatures. We have identified two human cDNAs encoding proteins which share sequence identity to the yeast CDC14p. The cell cycle arrest in cdc14-1(ts) can be specifically complemented by the human cDNAs suggesting that they are functionally equivalent to the yeast CDC14 gene. Another clone identified in the search for human CDC14-like proteins corresponded to the putative tumor suppressor gene PTEN/MMAC1 (phosphatase and tensin homologue deleted on chromosome 10 or mutated in multiple advanced cancers 1). Analysis of the PTEN/MMAC1 showed that it did not complement the cdc14-1(ts) allele and that it is more closely related to the yeast open reading frame YNL128W. Human CDC14p and PTEN/MMAC1 were expressed as recombinant proteins, and both were shown to have kinetic properties characteristic of dual specific phosphatases. The human CDC14p was localized in the nucleus while PTEN/MMAC1 has been reported to be localized in the cytoplasm. Our results suggest that CDC14 and YNL128W/PTEN/MMAC1 represent two related, but distinct, families of human and yeast phosphatases.
Assuntos
Proteínas de Ciclo Celular/química , Monoéster Fosfórico Hidrolases , Proteínas Tirosina Fosfatases/química , Proteínas de Saccharomyces cerevisiae , Proteínas Supressoras de Tumor , Alelos , Sequência de Aminoácidos , Sítios de Ligação , Compartimento Celular , Ciclo Celular , Proteínas de Ciclo Celular/genética , Sequência Conservada , DNA Complementar/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Cinética , Dados de Sequência Molecular , Miocárdio/química , Fases de Leitura Aberta , PTEN Fosfo-Hidrolase , Proteínas Tirosina Fosfatases/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Alinhamento de SequênciaRESUMO
The global regulator Lrp (leucine-responsive regulatory protein), in some cases modulated by its co-regulator leucine, has been shown to regulate more than 40 genes and operons in Escherichia coli. Leucine modulates Lrp regulation of leucine-responsive operons. The level of sensitivity of these operons to leucine varies greatly, but the basis for this variation is only partially understood. One operon controlled by Lrp that is relatively insensitive to leucine is gltBDF, which includes genes specifying the large (GltB) and small (GltD) subunits of glutamate synthase. Earlier gel mobility shift assays have demonstrated that Lrp binds to a fragment of DNA containing the gltBDF promoter region. To further define the nature of this Lrp-gltBDF interaction, DNase I footprinting experiments were performed. The results indicate that Lrp binds cooperatively to three sites quite far upstream, spanning the region from -140 to -260 base-pairs relative to the start of transcription. Phased hypersensitivity is observed throughout the entire binding region, suggesting that Lrp bends the DNA. To determine the relative importance of these three sites for the transcriptional activation of gltBDF, a series of site-directed mutations was generated. The effects of these mutations on Lrp binding were determined both by DNase I footprinting and by quantitative mobility shift assays, while their effects on transcription in vivo were examined by measuring beta-galactosidase activity levels of chromosomal gltB::lacZ operon fusions. Our results indicate that all three sites are required for maximal gene expression, as is the proper phasing of the sites with one another and with the start of transcription. Our results suggest that Lrp binds a central palindromic site, interacting predominantly with the major groove of its DNA target, and that additional dimers bind to flanking sites to form a nucleoprotein activation complex.
Assuntos
Proteínas de Bactérias/genética , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Glutamato Sintase/genética , Nucleoproteínas/genética , Óperon , Pegada de DNA , Desoxirribonuclease I/efeitos dos fármacos , Desoxirribonuclease I/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Leucina/genética , Leucina/farmacologia , Proteína Reguladora de Resposta a Leucina , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Fatores de Transcrição/genéticaRESUMO
We report the identification of the PPS1 gene of Saccharomyces cerevisiae. The deduced amino acid sequence of PPS1p shows similarity with protein-tyrosine phosphatases (PTPases) and is most closely related to a subfamily of PTPases that are capable of dephosphorylating phosphoseryl and phosphothreonyl residues as well as phosphotyrosyl residues. Analysis of the predicted amino acid sequence suggests that the protein consists of an active phosphatase domain, an inactive phosphatase-like domain, and an NH2-terminal extension. Mutation of the catalytic cysteinyl residue in the active phosphatase domain reduced the in vitro activity of the mutant protein to less than 0.5% of wild type activity, while mutation of the corresponding cysteinyl residue of the inactive phosphatase-like domain had no effect on in vitro activity. The PPS1 protein was expressed in Escherichia coli, and the protein was shown to catalyze the hydrolysis of p-nitrophenyl phosphate, dephosphorylate phosphotyrosyl, and phosphothreonyl residues in synthetic diphosphorylated peptides and to inactivate the human ERK1 protein. PPS1 transcript abundance is coregulated with that of the divergently transcribed DPB3 gene, which codes for a subunit of DNA polymerase II, with both transcripts showing peak abundance in S phase. pps1Delta mutant strains did not differ from PPS1 strains under any of the conditions tested, but overexpression of the PPS1 protein in S. cerevisiae led to synchronous growth arrest and to aberrant DNA synthesis. A screen for suppressors of this growth arrest identified the RAS2 gene as a multicopy suppressor of the PPS1p overexpression arrest. The arrest was not suppressed by the presence of multicopy RAS1, TPK2, or TPK3 genes or by the presence of 5 mM cAMP in the growth medium, suggesting that PPS1 functions in a pathway involving RAS2, but not TPK kinases or adenylate cyclase.
Assuntos
Proteínas de Bactérias/genética , Replicação do DNA , Genes Fúngicos , Isoenzimas/genética , Proteínas Tirosina Fosfatases/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Ciclo Celular , DNA Fúngico/biossíntese , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Isoenzimas/química , Dados de Sequência Molecular , Proteínas Tirosina Fosfatases/química , Saccharomyces cerevisiae/enzimologia , Transcrição Gênica , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
The two major porins of Escherichia coli K-12 strains, OmpC and OmpF, are inversely regulated with respect to one another. The expression of OmpC and OmpF has been shown to be influenced by the leucine-responsive regulatory protein (Lrp): two-dimensional gel electrophoresis of proteins from strains with and strains without a functional Lrp protein revealed that OmpC expression is increased in an lrp strain, while OmpF expression is decreased. In agreement with these findings, we now present evidence that transcriptional (operon) fusions of lacZ+ to ompC and micF are negatively regulated by Lrp. Lrp binds specifically to the intergenic region between micF and ompC, as indicated by mobility shift assays and by DNase I footprinting. The expression of an ompF'-lacZ+ gene (translational) fusion is increased 3.7-fold in an lrp+ background compared with an lrp background, but expression of an ompF-lacZ+ operon fusion is not. Studies of in vivo expression of the outer membrane porins during growth on glucose minimal medium showed that the OmpF/OmpC ratio is higher in lrp+ strains than it is in isogenic lrp strains. The effect of Lrp was not seen in a strain containing a deletion of micF. Our studies suggest that the positive effect of Lrp on OmpF expression stems from a negative effect of Lrp on the expression of micF, an antisense RNA that inhibits ompF translation.
Assuntos
Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição , Proteínas da Membrana Bacteriana Externa/genética , Sequência de Bases , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli , Proteína Reguladora de Resposta a Leucina , Dados de Sequência Molecular , Pressão Osmótica , Plasmídeos/genética , Porinas/biossíntese , Ligação Proteica , Biossíntese de Proteínas , Proteínas Recombinantes de Fusão/biossíntese , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Gênica , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
The regulon controlled by the leucine-responsive regulatory protein (Lrp) of Escherichia coli consists of over 40 genes and proteins whose expression is regulated, either positively or negatively, by Lrp. The gltBDF operon, encoding glutamate synthase, was originally identified as a member of the Lrp regulon through a two-dimensional electrophoretic analysis of polypeptides from isogenic strains containing or lacking a functional Lrp protein. We have now demonstrated that Lrp regulates the transcription of gltBDF::lacZ operon fusions. Relative to expression in glucose minimal 3-(N-morpholino)propanesulfonic acid (MOPS) medium, gltBDF::lacZ expression in an lrp+ strain is repressed 2.2-fold in the presence of 10 mM exogenous leucine and 16-fold in Luria broth. Repression of gltBDF::lacZ expression by leucine or Luria broth is not seen for an isogenic strain containing a Tn10 insertion in lrp, and expression of gltBDF::lacZ is 44-fold lower than in the lrp+ strain when both are grown in glucose minimal MOPS medium. Lrp binds specifically to DNA fragments containing the gltBDF promoter region. Saturating levels of leucine do not abolish binding of Lrp upstream of gltBDF but merely increase its apparent dissociation constant from 2.0 to 6.9 nM. Electrophoretic analysis of the Lrp regulon established that target proteins differ greatly in the degree to which the effect of Lrp on their expression is antagonized by leucine. On the basis of our present results, we present a model for positive regulation of target genes by Lrp. Insensitivity to leucine would be expected when the effective intracellular concentration of Lrp is high relative to the affinity of Lrp binding sites required for transcription of the target gene. At lower concentrations of Lrp, transcription of the target gene should be sensitive to leucine. This model suggests that regulation of the concentration of active Lrp is critical to control of the Lrp regulon.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Glutamato-Amônia Ligase/biossíntese , Leucina/farmacologia , Óperon , Fatores de Transcrição , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Primers do DNA , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Glutamato-Amônia Ligase/genética , Cinética , Leucina/metabolismo , Proteína Reguladora de Resposta a Leucina , Dados de Sequência Molecular , Óperon/efeitos dos fármacos , Plasmídeos , Regiões Promotoras Genéticas , Ligação Proteica , Mapeamento por Restrição , Transcrição Gênica , beta-Galactosidase/biossíntese , beta-Galactosidase/metabolismoRESUMO
In Escherichia coli K-12, expression of the lysU gene is regulated by the lrp gene product, as indicated by an increase in the level of lysyl-tRNA synthetase activity and LysU protein in an lrp mutant. Comparison of the patterns of protein expression visualized by two-dimensional gel electrophoresis indicated that LysU is present at higher levels in an lrp strain than in its isogenic lrp+ parent. The purified lrp gene product was shown to bind to sites upstream of the lysU gene and to protect several sites against DNase I digestion. A region extending over 100 nucleotides, between 60 and 160 nucleotides upstream from the start of the lysU coding sequence, showed altered sensitivity to DNase I digestion in the presence of the Lrp protein. The extent of protected DNA suggests a complex interaction of Lrp protein and upstream lysU DNA.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Lisina-tRNA Ligase/genética , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/genética , Proteínas de Escherichia coli , Proteína Reguladora de Resposta a Leucina , Dados de Sequência Molecular , Mutagênese , Biossíntese de ProteínasRESUMO
The leucine-responsive regulatory protein (Lrp) has been shown to regulate, either positively or negatively, the transcription of several Escherichia coli genes in response to leucine. We have used two-dimensional gel electrophoresis to analyze the patterns of polypeptide expression in isogenic lrp+ and lrp mutant strains in the presence or absence of leucine. The absence of a functional Lrp protein alters the expression of at least 30 polypeptides. The expression of the majority of these polypeptides is not affected by the presence or absence of 10 mM exogenous leucine. Outer membrane porins OmpC and OmpF, glutamine synthetase (GlnA), the small subunit of glutamate synthase (GltD), lysyl-tRNA synthetase form II (LysU), a high-affinity periplasmic binding protein specific for branched-chain amino acids (LivJ), W protein, and the enzymes of the pathway converting threonine to glycine, namely, threonine dehydrogenase (Tdh) and 2-amino-3-ketobutyrate coenzyme A ligase (Kbl), were identified as members of the Lrp regulon by electrophoretic analysis. We have shown that Lrp is a positive regulator of glutamate synthase and glutamine synthetase and that exogenous leucine has little or no effect on the expression of these proteins. In strains carrying a glnL deletion and in strains carrying the glnL2302 allele, which directs the synthesis of a GlnL protein that is constitutively active, expression of glutamine synthetase is no longer regulated by Lrp, demonstrating that the effect of Lrp on glutamine synthetase levels is indirect and requires an intact glnL gene. lrp::Tn10 strains grow poorly when arginine or ornithine is present as the sole nitrogen source in the medium. On the bases of present studies and previous research, we propose that Lrp is involved in the adaptation of E. coli cells to major shifts in environment, such as those which occur when E. coli leaves the intestinal tract of its animal host. Several genes required for amino acid and peptide transport and catabolism are negatively regulated by Lrp, and other genes required for amino acid biosynthesis and ammonia assimilation in a nitrogen-poor environment are positively regulated by Lrp.