RESUMEN
We have isolated a new type of ATP-dependent protease from Escherichia coli. It is the product of the heat-shock locus hslVU that encodes two proteins: HslV, a 19-kDa protein similar to proteasome beta subunits, and HslU, a 50-kDa protein related to the ATPase ClpX. In the presence of ATP, the protease hydrolyzes rapidly the fluorogenic peptide Z-Gly-Gly-Leu-AMC and very slowly certain other chymotrypsin substrates. This activity increased 10-fold in E. coli expressing heat-shock proteins constitutively and 100-fold in cells expressing HslV and HslU from a high copy plasmid. Although HslV and HslU could be coimmunoprecipitated from cell extracts of both strains with an anti-HslV antibody, these two components were readily separated by various types of chromatography. ATP stimulated peptidase activity up to 150-fold, whereas other nucleoside triphosphates, a nonhydrolyzable ATP analog, ADP, or AMP had no effect. Peptidase activity was blocked by the anti-HslV antibody and by several types of inhibitors of the eukaryotic proteasome (a threonine protease) but not by inhibitors of other classes of proteases. Unlike eukaryotic proteasomes, the HslVU protease lacked tryptic-like and peptidyl-glutamyl-peptidase activities. Electron micrographs reveal ring-shaped particles similar to en face images of the 20S proteasome or the ClpAP protease. Thus, HslV and HslU appear to form a complex in which ATP hydrolysis by HslU is essential for peptide hydrolysis by the proteasome-like component HslV.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Endopeptidasas/metabolismo , Escherichia coli/enzimología , Proteínas de Choque Térmico/metabolismo , Serina Endopeptidasas/metabolismo , Proteasas ATP-Dependientes , Secuencia de Aminoácidos , Hidrólisis , Microscopía Electrónica , Datos de Secuencia Molecular , Especificidad por SustratoAsunto(s)
Proteínas de Escherichia coli , Proteínas de Choque Térmico/metabolismo , Proteasa La , Serina Endopeptidasas/metabolismo , Proteasas ATP-Dependientes , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Escherichia coli/enzimología , Proteínas de Choque Térmico/aislamiento & purificación , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/aislamiento & purificaciónRESUMEN
Factors influencing the direct transformation of the yeast Saccharomyces cerevisiae with synthetic oligonucleotides were investigated by selecting for cyc1 transformants that contained at least partially functional iso-1-cytochrome c. Approximately 3 x 10(4) transformants, constituting 0.1% of the cells, were obtained by using 1 mg of oligonucleotide in the reaction mixture. Carrier, such as heterogeneous oligonucleotides, enhanced transformation frequencies. Transformation frequencies were dramatically reduced if the oligonucleotides had a large number of mismatches or had terminally located mismatches. Transformation with oligonucleotides, but not with linearized double-strand plasmid, was efficient in a rad52- strain, suggesting that the pathway for transformation with oligonucleotides is different from that with linearized double-strand plasmid. We describe a procedure of co-transformation with two oligonucleotides, one correcting the cyc1 defect of the target allele in the host strain, and the other producing a desired amino acid alteration elsewhere in the iso-1-cytochrome c molecule; approximately 20% of the transformants obtained by co-transformation contained these desired second alterations.
Asunto(s)
Citocromos c , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transformación Genética , Secuencia de Aminoácidos , Secuencia de Bases , Grupo Citocromo c/genética , ADN/farmacología , Reparación del ADN/genética , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Oligodesoxirribonucleótidos/síntesis química , ARN de Transferencia/farmacología , Transformación Genética/efectos de los fármacosRESUMEN
Cytochrome c with a nuclear localization signal added at the N terminus was mistargeted to the nucleus, resulting in a yeast strain deficient in mitochondrial cytochrome c. Reversion of this strain allowed the isolation of temperature-conditional mutants defective in nuclear transport, as demonstrated with one of these mutants, nip1-1, that was shown to be defective in nuclear accumulation of a LacZ protein containing a nuclear localization signal of the yeast ribosomal protein L29. The NIP1+ gene was cloned and shown to encode a 93,143-Da protein. Furthermore, an epitope-labeled NIP1 protein migrated in SDS/polyacrylamide gels with a mass of approximately 100,000 Da and was shown by immunofluorescence to localize mainly in the cytoplasm. NIP1+ was shown to be an essential gene by gene disruption experiments. Intriguingly, NIP1 has a serine-rich acidic N-terminal region that is similar in this regard to the N-terminal region of a previously described nuclear localization signal-binding protein, NSR1.
Asunto(s)
Núcleo Celular/metabolismo , Grupo Citocromo c/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Codón/genética , Factor 3 de Iniciación Eucariótica , Proteínas Fúngicas/aislamiento & purificación , Prueba de Complementación Genética , Immunoblotting , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/aislamiento & purificación , Fenotipo , Biosíntesis de Proteínas , Proteínas Recombinantes de Fusión/aislamiento & purificación , Saccharomyces cerevisiae/metabolismo , TATA Box , beta-Galactosidasa/genética , beta-Galactosidasa/aislamiento & purificaciónRESUMEN
Cyc1 mutants of the yeast Saccharomyces cerevisiae were directly transformed with both sense and antisense oligonucleotides to examine the involvement of the two genomic DNA strands in transformation. Sense oligonucleotides yielded approximately 20-fold more transformants than antisense oligonucleotides. This differential effect was observed with oligonucleotides designed to make alterations at six different sites along the gene and was independent of the oligonucleotide sequence and length, number of mismatches and the host strain. Competition studies showed that antisense oligonucleotides did not inhibit transformation. Although the mechanism for this strand specificity is unknown, this difference was maintained even when CYC1 transcription was diminished to approximately 2% of the normal level.
Asunto(s)
Citocromos c , Oligodesoxirribonucleótidos/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transformación Genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Grupo Citocromo c/genética , ADN sin Sentido/genética , ADN de Hongos/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos/síntesis química , Saccharomyces cerevisiae/enzimología , Transcripción GenéticaAsunto(s)
Citocromos c , Mutagénesis Sitio-Dirigida , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Bases , Grupo Citocromo c/genética , Mutación del Sistema de Lectura , Genes Fúngicos , Técnicas Genéticas , Datos de Secuencia Molecular , Sondas de OligonucleótidosRESUMEN
The specificities of methionine aminopeptidase and amino-terminal acetylation in the yeast Saccharomyces cerevisiae were investigated in vivo by sequencing a series of altered iso-1-cytochrome c. Twenty iso-1-cytochromes c, each having a different penultimate residue in the sequence Met-Xaa-Phe-Leu-, were created by transforming yeast directly with synthetic oligonucleotides. The degree of methionine cleavage and amino-terminal acetylation was estimated from the levels of pertinent peptides separated by high performance liquid chromatography. The results confirmed our earlier hypothesis (Sherman, F., Stewart, J. W., and Tsunasawa, S. (1985) BioEssays 3, 27-31) that methionine is completely removed from penultimate residues having radii of gyration of 1.29 A or less (glycine, alanine, serine, cysteine, threonine, proline, and valine). However, only partial cleavage occurred in the sequences Met-Thr-Pro-Leu- and Met-Val-Pro-Leu-, demonstrating that proline at the third position inhibits methionine cleavage when the penultimate residue has an intermediate radius of gyration. Acetylation of the retained amino-terminal methionine occurred completely with the Ac-Met-Glu-Phe-Leu- and Ac-Met-Asp-Phe-Leu- sequences and partially with the Ac-Met-Asn-Phe-Leu-sequence. Although the consensus for acetylation of the retained amino-terminal methionine is not completely known, these results and the results of published sequences indicated that Ac-Met-Glu- and Ac-Met-Asp- (methionine followed by an acidic residue) is sufficient for amino-terminal acetylation in eukaryotes but not in prokaryotes.
Asunto(s)
Aminopeptidasas/metabolismo , Grupo Citocromo c/metabolismo , Citocromos c , Metionina/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Acetilación , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Grupo Citocromo c/química , Grupo Citocromo c/genética , Metionil Aminopeptidasas , Datos de Secuencia Molecular , Especificidad por Sustrato , Transformación GenéticaRESUMEN
A gene from Saccharomyces cerevisiae has been mapped, cloned, sequenced and shown to encode a catalytic subunit of an N-terminal acetyltransferase. Regions of this gene, NAT1, and the chloramphenicol acetyltransferase genes of bacteria have limited but significant homology. A nat1 null mutant is viable but exhibits a variety of phenotypes, including reduced acetyltransferase activity, derepression of a silent mating type locus (HML) and failure to enter G0. All these phenotypes are identical to those of a previously characterized mutant, ard1. NAT1 and ARD1 are distinct genes that encode proteins with no obvious similarity. Concomitant overexpression of both NAT1 and ARD1 in yeast causes a 20-fold increase in acetyltransferase activity in vitro, whereas overexpression of either NAT1 or ARD1 alone does not raise activity over basal levels. A functional iso-1-cytochrome c protein, which is N-terminally acetylated in a NAT1 strain, is not acetylated in an isogenic nat1 mutant. At least 20 other yeast proteins, including histone H2B, are not N-terminally acetylated in either nat1 or ard1 mutants. These results suggest that NAT1 and ARD1 proteins function together to catalyze the N-terminal acetylation of a subset of yeast proteins.
Asunto(s)
Acetiltransferasas/genética , Citocromos c , Genes Fúngicos , Genes del Tipo Sexual de los Hongos , Genes , Mutación , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Grupo Citocromo c/genética , Histonas/genética , Datos de Secuencia Molecular , Acetiltransferasa A N-Terminal , Fenotipo , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Ácido NucleicoRESUMEN
Genomic DNA of the yeast, Saccharomyces cerevisiae, can be conveniently and specifically altered by transforming spheroplasts or lithium acetate-treated cells directly with synthetic oligonucleotides. Altered forms of iso-1-cytochrome c were generated by transforming a cyc1 mutant with oligonucleotides and selecting for at least partially functional revertants; the oligonucleotides contained a sequence that corrected the cyc1 mutation and produced additional alterations at nearby sites. Transformation has been accomplished with oligonucleotides as short as 20 nucleotides and with amounts as low as 100 micrograms. This method of site-directed mutagenesis in vivo has been used to produce alterations in the NH2-terminal region of iso-1-cytochrome c in which the NH2-terminal methionine is excised and the penultimate residue is acetylated.