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D J Ecker

Publications and source records attributed to D J Ecker.

54 records · Page 3Linked to original sources
PubMed1989

Ubiquitin function studied by disulfide engineering.

Disulfide engineering was used to probe the role of conformational mobility in ubiquitin-mediated proteolysis. Six genes that encode cysteine-containing mutants of ubiquitin were constructed, expressed in Escherichia coli and the proteins purified. Single cysteine-containing mutants and a 4/14 disulfide were active in degradation of a substrate protein in vitro, while the 4/66 disulfide, which cross-links the NH2- and COOH-terminal strands of the protein, was only 20-30% active. The solution structure of the 4/66 mutant was solved: the disulfide is left-handed with no perturbations in the backbone from that of wild type ubiquitin. The results suggest that conformational mobility is required for the activity of ubiquitin in signaling proteolysis.

Computer Simulation↗
PubMed1989

Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli.

Despite the availability of efficient transcription and translation signals, some heterologous gene products are not adequately expressed when introduced into prokaryotes and eukaryotes. An expression system has been established in Escherichia coli to increase the yield of cloned gene products, where the C terminus of ubiquitin was fused to the N terminus of unstable or poorly expressed proteins. Fusion of ubiquitin to yeast metallothionein or to the alpha subunit of the adenylate cyclase-stimulatory GTP-binding protein increased the yield from undetectable to 20% of the total cellular protein. A ubiquitin-N alpha-protein hydrolase has been partially purified from rabbit reticulocytes; this enzyme faithfully cleaves the junction peptide bound between the C-terminal Gly-76 of ubiquitin and the fusion protein. The increased yield of cloned gene products is very likely due to increased stability and/or more efficient translation of the fusion proteins. Possible mechanisms for the augmentation of ubiquitin fusion-protein expression in prokaryotes and eukaryotes are discussed.

Cloning, Molecular↗
PubMed1988

Ubiquitin-metallothionein fusion protein expression in yeast. A genetic approach for analysis of ubiquitin functions.

We have established a Saccharomyces cerevisiae genetic system that expresses the fusion protein ubiquitin-metallothionein. We have evaluated the effects of amino-terminal ubiquitination of metallothionein on the stability and function of metallothionein. The fusion protein of wild type ubiquitin and metallothionein was rapidly processed in vivo to release free ubiquitin and metallothionein. Site-directed mutants of ubiquitin-metallothionein expressed in yeast were used to study the specificity of the (alpha-NH2-ubiquitin) protein endopeptidases. The data suggest that amino-terminal ubiquitination is not a signal for the proteolysis of yeast metallothionein in yeast. We have also discovered that expression of selected ubiquitin mutants blocked the growth of yeast. The data suggest that in addition to its function as a proteolytic signal, ubiquitination of proteins plays multiple roles in the cell.

Copper↗
PubMed1988

Ubiquitin carboxyl-terminal peptides. Substrates for ubiquitin activating enzyme.

The carboxyl terminus of ubiquitin is activated in the presence of ATP to enter the ubiquitin cycle in cells. Peptides corresponding to the COOH-terminal region of ubiquitin were synthesized to investigate their effects on the ATP/ubiquitin-dependent proteolytic pathway. Their activities in the PPi exchange assay with ubiquitin activating enzyme (E1) were proportional to their length. The hexapeptide Ac-Leu-Arg-Leu-Arg-Gly-Gly reacted with ATP to form an enzyme-adenylate-hexapeptide complex and at high concentrations was 20-25% as active as human ubiquitin in the PPi exchange assay with E1. However, the hexapeptide was not transferred to the sulfhydryl "thiol" site on E1. In addition, the COOH-terminal peptides did not support the degradation of 125I-bovine serum albumin in the reticulocyte lysate system. A nonhomologous peptide of equivalent length was inactive in all assays. Thus, synthetic COOH-terminal peptide(s) of ubiquitin can partially substitute for ubiquitin in its reactions with E1 but do not support subsequent steps of the energy-dependent proteolytic pathway. These results show that it may be possible to design small molecules that either serve as substrates or inhibitors for other specific steps in ubiquitin-dependent pathways.

Adenosine Triphosphate↗
PubMed1987

Expression and accurate processing of yeast penta-ubiquitin in Escherichia coli.

An expression vector (pSJyub-5) was constructed which contained five repeats of the "yeast ubiquitin gene" regulated by a heat-inducible lambda PL promoter. The vector, when expressed in Escherichia coli, produced a penta-ubiquitin of approximately 42 kDa. Purified penta-ubiquitin was found to be as active as the human mono-ubiquitin in the in vitro ATP/ubiquitin-dependent proteolytic assay of the reticulocyte lysate, indicating that the expressed gene product was recognized by the enzymes involved in the ATP/ubiquitin-dependent proteolytic pathway. The inability of penta-ubiquitin to act as a substrate in the pyrophosphate exchange reaction with the ubiquitin-activating enzyme E1 suggested that it had a carboxyl-terminal Asn, in agreement with the nucleotide sequence. In E. coli, the expressed penta-ubiquitin was processed correctly to mono-ubiquitin. The fidelity of processing in E. coli was confirmed by the following observations. The amino acid compositions of the processed mono-ubiquitin and human ubiquitin were similar. The 1H NMR spectrum of peaks representing amide hydrogens of the carboxyl-terminal Arg-74, Gly-75, and Gly-76 of the processed mono-ubiquitin was identical with that of human ubiquitin. The immunoreactivity of processed mono-ubiquitin and human ubiquitin against polyclonal antibodies that recognized epitope(s) only on the carboxyl terminus of ubiquitin were similar. The human and processed mono-ubiquitin were equally active in degrading 125I-bovine serum albumin in the ATP/ubiquitin-dependent in vitro proteolytic assay with reticulocyte lysates. In the pyrophosphate exchange assay with isolated ubiquitin activating enzyme E1, they were also equally reactive, confirming that the expressed and processed ubiquitin contained an intact carboxyl-terminal Gly-76. Purified penta-ubiquitin should prove to be a useful substrate for identifying and isolating processing enzyme(s) involved in the ATP/ubiquitin-dependent proteolytic pathway.

Amino Acids↗
PubMed1987

Gene synthesis, expression, structures, and functional activities of site-specific mutants of ubiquitin.

To study the structure and function of ubiquitin we have chemically synthesized a ubiquitin gene that encodes the amino acid sequence of animal ubiquitin, inserting a series of restriction enzyme sites that divide the gene into eight "mutagenesis modules." A series of site-specific mutations were constructed to selectively perturb various regions of the molecule. The mutant genes were expressed in a large quantity of Escherichia coli, and the modified proteins were purified. To determine the structural effects of the amino acid substitutions, the solution structure of ubiquitin was investigated by two-dimensional NMR and each of the mutant proteins were screened for structural perturbations. With one exception, virtually no changes were seen other than at the point of mutation. Functional studies of the mutant proteins with the ubiquitin-activating enzyme E1 and in the reticulocyte protein degradation assay were used to identify regions of the molecule important to ubiquitin's activity in intracellular proteolysis.

Amino Acid Sequence↗
PubMed1987

Chemical synthesis and expression of a cassette adapted ubiquitin gene.

A gene encoding the yeast ubiquitin was chemically synthesized and expressed in yeast under regulatory control of the copper metallothionein (CUP1) promoter. The gene was assembled in a one-step ligation reaction from eight oligonucleotide fragments ranging in length from 50 to 64 nucleotides. To facilitate mutagenesis and gene fusion studies, eight unique 6-base-cutting restriction enzyme sites were placed in the reading frame which did not alter the encoded protein sequence or force the utilization of rare codons. In a copper-resistant yeast strain (CUP1r), expression of the gene was induced by copper to approximately 5% of the total yeast proteins, as determined by Coomassie-stained polyacrylamide gels. The protein, purified from yeast, reacted with ubiquitin-specific antibodies and was found to be biologically active in supporting ubiquitin-dependent protein degradation in vitro.

Amino Acid Sequence↗
PubMed1987

A vector for construction of gene libraries and the expression of heterologous genes in Saccharomyces cerevisiae.

We have constructed a convenient new vector, YEp-DE, for the construction of gene libraries and the expression of heterologous genes in Saccharomyces cerevisiae. The vector contains the yeast LEU2 gene, the 2 mu origin of replication, and a region from pUC18 that includes the ampr gene, the Escherichia coli origin of replication (ori), and the LacZ gene with multiple cloning sites. Five sites (Sac1, Sma1, BamH1, Sal1, Sph1) in this region are unique. This vector has advantages over similar yeast-E. coli shuttle vectors: small size (7291 bp, entirely sequenced), convenient cloning sites, and lacZ selection for detecting recombinant plasmids.

Cloning, Molecular↗
PubMed1987

Induction of metallothionein is correlated with resistance to auranofin, a gold compound, in Chinese hamster ovary cells.

Metallothioneins (MTs) are low molecular weight, thiol-rich, metal-binding proteins. Auranofin (AF) is a gold compound active in the treatment of rheumatoid arthritis. The effects of AF on regulation of MT gene expression in Chinese hamster ovary cells were studied. AF-resistant cells accumulated substantial amounts of MT mRNA and protein, whereas no induction was observed in AF-sensitive cells. Cells capable of inducing MT in the presence of AF were much less sensitive to AF-mediated cytotoxicity. Induction of MT by low concentrations of Cd protected cells from subsequently administered doses of AF. The level of protection correlated with the level of induced MT. These findings indicate that MT plays a central role in the mechanisms underlying cellular resistance to gold compounds.

Animals↗
PubMed1986

Yeast metallothionein function in metal ion detoxification.

A genetic approach was taken to test the function of yeast metallothionein in metal ion detoxification. A yeast strain was constructed in which the metallothionein locus was deleted (cup1 delta). The cup1 delta strain was complemented with normal or mutant metallothionein genes under normal or constitutive regulatory control on high copy episomal plasmids. Metal resistance of the cup1 delta strain with and without the metallothionein-expressing vectors was analyzed. The normally regulated metallothionein gene conferred resistance only to copper (1000-fold); constitutively expressed metallothionein conferred resistance to both copper (500-fold) and cadmium (1000-fold), but not to mercury, zinc, silver, cobalt, nickel, gold, platinum, lanthanum, uranium, or tin. Two mutant versions of the metallothionein gene were constructed and tested for their ability to confer metal resistance in the cup1 delta background. The first had a deletion of a highly conserved amino acid sequence (Lys-Lys-Ser-Cys-Cys-Ser). The second was a hybrid gene consisting of the sequences coding for the first 20 amino acids of the yeast protein fused to the monkey metallothionein gene. Expression of these genes under the CUP1 promoter provided significant protection from copper, but none of the other metals tested. These results demonstrate that there is significant flexibility in the structural requirements for metallothionein to function in copper detoxification and that yeast metallothionein is also capable of detoxifying cadmium under conditions of constitutive expression.

Amino Acid Sequence↗
PubMed1986

Metallothionein turnover in mammalian cells. Implications in metal toxicity.

Metallothioneins are low molecular weight, cysteine-rich proteins believed to participate in metal detoxification. Turnover of Cd-, Zn-, and Au-induced metallothionein was studied in a Chinese hamster ovary cell line which was resistant to Cd and the Au-containing drug auranofin. Cd, Zn, and Au were potent inducers of metallothionein mRNA and resulted in accumulation of approximately equal amounts of mRNA. Pulse-chase studies with [35S]cysteine revealed that the half-life of Au-, Zn-, and Cd-induced metallothionein was 0.75, 10, and 24 h, respectively. The differences in the half-life of metallothionein may be related to the tertiary structure of metal-metallothionein complexes. These results have implications in the mechanism of resistance to gold compounds.

Animals↗
PubMed1986

Recognition and transport of ferric enterobactin in Escherichia coli.

The specificity of the outer membrane protein receptor for ferric enterobactin transport in Escherichia coli and the mechanism of enterobactin-mediated transport of ferric ions across the outer membrane have been studied. Transport kinetic and inhibition studies with ferric enterobactin and synthetic structural analogs have mapped the parts of the molecule important for receptor binding. The ferric complex of the synthetic structural analog of enterobactin, 1,3,5-N,N',N''-tris-(2,3-dihydroxybenzoyl)triaminomethylbenzene (MECAM), was transported with the same maximum velocity as was ferric enterobactin. A double-label transport assay with [59Fe, 3H]MECAM showed that the ligand and the metal are transported across the outer membrane at an identical rate. Under the growth conditions used, large fractions of the transported complexes were available for exchange across the outer membrane when a large excess of extracellular complex was added to the cell suspension; at least 60% of the internalized [59Fe]enterobactin exchanged with extracellular [55Fe]enterobactin. Internalized [59Fe, 3H]MECAM was released from the cell as the intact complex when either unlabeled Fe-MECAM or Fe-enterobactin was added extracellularly. The results suggest a mechanism of active transport of unmodified coordination complex across the outer membrane with possible accumulation in the periplasm.

Bacterial Outer Membrane Proteins↗
PubMed1986

Escherichia coli iron enterobactin uptake monitored by Mössbauer spectroscopy.

Iron uptake by Escherichia coli under aerobic conditions of iron deficiency is mediated by a highly stable ferric enterobactin [Fe(ent)3-] siderophore complex. Mössbauer spectroscopy has been used to monitor the fate of the iron as 57Fe(ent) was taken up by the cells. Osmotic shock experiments were used to distinguish between the iron present in the periplasmic space and that in the cytoplasm of the cell. Iron delivery by a synthetic analog of enterobactin, 1,3,5-N,N',N''- tris-(2,3-dihydroxybenzoyl)triaminomethylbenzene (MECAM), was also studied. Although Fe-MECAM was transported at the same rate as was Fe(ent) across the outer membrane and was apparently accumulated in the periplasmic space, the subsequent behaviors of Fe(ent) and Fe-MECAM were very different. After more than 30 min, a major fraction of the iron originally absorbed as ferric enterobactin appeared as Fe(II), apparently in the cytoplasm of the cell. However, little iron was delivered to the cytoplasm by the MECAM complex. The differences in specificity of these two stages of iron uptake by E. coli are discussed.

Bacterial Outer Membrane Proteins↗
PubMed1983

Iron uptake from ferrichrome A and iron citrate in Ustilago sphaerogena.

Double radioactive label transport assays with iron, chromium, and gallium chelates were used to investigate the mechanism of iron uptake by Ustilago sphaerogena. In iron-deficient cells, ferrichrome A iron was taken up without appreciable uptake of the ligand. Iron-sufficient cells partially accumulated the ligand with the metal. The chromium- and gallium-containing analogs of ferrichrome A were transported as intact chelates. Ferrichrome A iron uptake was inhibited by dipyridyl. The data suggest that the intact ferrichrome A chelate binds to a specific receptor, the iron is then separated from the ligand at the membrane by reduction, and the metal is released to the inside of the cell while the ligand is released to the exterior. The reduction step is not transport rate limiting. Iron chelated to citrate was taken up by an energy-dependent process. The citrate ligand was not taken up with the metal. Uptake was sensitive to dipyridyl and ferrozine. Chromic ion chelated to citrate was not transported, suggesting that the iron, rather than the chelate, is recognized by the receptor or that reduction of the metal is required for transport.

Basidiomycota↗
PubMed1982

Siderophore iron transport followed by electron paramagnetic resonance spectroscopy.

Siderophore iron transport was followed in Ustilago sphaerogena using isotope transport assays coupled with EPR spectroscopy. EPR spectroscopy was used as a quantitative tool to follow the rate of reduction of siderophore iron(III) to iron(II) in the cell suspension by following the disappearance of the signal at g = 4.3. This rate was compared with the rate of iron transport, measured by the disappearance of radioactively labeled iron from the medium. The transport of three iron chelates was examined: the ferric siderophores ferrichrome and ferichrome A, and iron(III) chelated to excess citrate. For the transport of ferrichrome, an iron(III) ionophore, the rate of reduction of iron(III) to iron(II) was significantly lower than the rate of uptake of isotope from the medium supernatant, which is consistent with the established mechanism of uptake of the entire complex followed by intracellular reduction to remove the iron from the ligand. However, the rate of reduction of ferrichrome A, a non-ionophore, was identical with the rate of transport of iron into the cell. Iron(III) citrate was reduced at a rate slightly lower than the rate of transport. These data suggest that reduction of iron(III) is involved in the transport of iron from ferichrome A and possibly from iron(III) citrate.

Biological Transport, Active↗
PubMed1982

Role of two siderophores in Ustilago sphaerogena. Regulation of biosynthesis and uptake mechanisms.

Under iron-deficient conditions the smut fungus Ustilago sphaerogena produces two kinds of siderophores, ferrichrome and ferrichrome A. Regulation of ligand biosyntheses and uptake mechanisms of the iron chelates were studied to determine the role of each chelate in U. sphaerogena. The biosynthesis of each ligand was differentially regulated. Ferrichrome A, the more effective chelate, was preferentially synthesized under more extreme conditions of iron stress, but completely repressed when the cell was supplied with sufficient iron. In contrast, biosynthesis of ferrichrome was strongly but not completely repressed by iron. The mechanism of repression was examined using a newly developed in vivo synthesis assay. Chromium and gallium-containing siderophore analogs had no effect on siderophore ligand biosynthesis. Iron, added as siderophores, resulted in increased oxygen uptake and amino acid transport, which was soon followed by decreased ligand biosynthesis, suggesting that regulation may be indirect and related to oxidative metabolism. Uptake experiments were used to rule out a ligand-exchange mechanism for ferrichrome A-iron transport. The data suggest that ferrichrome A-iron is taken up at a specific site that results in a rapid distribution of iron inside the cell.

Amino Acids↗