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M Inouye

Publications and source records attributed to M Inouye.

At least 361 records · Page 20Linked to original sources
PubMed1987

Localization and membrane topology of EnvZ, a protein involved in osmoregulation of OmpF and OmpC in Escherichia coli.

OmpR and EnvZ, the protein products of the ompB locus, are regulatory components required for osmoexpression of outer membrane porin proteins, OmpF and OmpC, in Escherichia coli. EnvZ is considered to be an osmosensor which transmits signals across the membrane to OmpR, a transcriptional activator for ompF and ompC. We inserted the envZ gene into a high expression vector, pIN-III. Following cellular fractionation, EnvZ was found to be localized in the inner membrane. Sequence analysis revealed that the signal peptide-like N-terminal sequence was not removed from the purified EnvZ. A genetic approach using EnvZ/beta-lactamase fusion proteins was taken to determine the topology of EnvZ in the inner membrane. When beta-lactamase was fused after the N-terminal signal peptide-like sequence, ampicillin resistance, conferred by the beta-lactamase moiety of the fusion protein, was expressed. However, when beta-lactamase was fused after the second downstream apolar sequence, the cells showed very poor ampicillin resistance indicating that the enzyme was localized on the cytoplasmic side of the inner membrane. The results of this approach reveal that the hydrophilic region of EnvZ between the two apolar sequences is periplasmically localized and that the hydrophilic region downstream of the second apolar sequence is cytoplasmically directed. These results were confirmed by partial proteolysis of the fusion proteins in intact cells.

Amino Acid Sequence↗
PubMed1987

Export and secretion of overproduced OmpA-beta-lactamase in Escherichia coli.

The export of beta-lactamase to the periplasm of Escherichia coli can be directed by the OmpA signal peptide in the secretion cloning vector pIN-III. The overproduction of the hybrid precursor specifically induces a delay in the onset of processing of newly synthesized polypeptide chains. However, when the processing starts, no alteration in the rate of cleavage itself is observed. Our results suggest that the temporal mode of processing (which reflects translocation) does not depend on the nature of the signal peptide but rather depends on the nature of the polypeptide chain exported.

Bacterial Outer Membrane Proteins↗
PubMed1987

Wild type and mutant signal peptides of Escherichia coli outer membrane lipoprotein interact with equal efficiency with mammalian signal recognition particle.

The signal peptide of the outer membrane lipoprotein (OMLP) of Escherichia coli was shown to be capable of promoting protein translocation across mammalian microsomal membranes in vitro. We assayed translocation of a fusion protein containing the OMLP signal peptide and nine amino acids of OMLP fused in frame to beta-lactamase. The efficiency with which the mammalian translocation machinery recognizes and accepts the OMLP signal peptide as substrate is indistinguishable from that of mammalian secretory proteins. Upon translocation mammalian signal peptidase processes the pre-OMLP-beta-lactamase protein at different sites than are utilized in vivo by E. coli OMLP signal peptidase (signal peptidase II) but that can be predicted as mammalian signal peptidase cleavage sites. Mutants in the OMLP signal peptide were tested for their ability to promote translocation of the fusion protein in this assay system. It has been shown previously that mutants in the positively charged amino acids at the amino terminus of the signal peptide severely delay the translocation of OMLP in vivo in E. coli. However, these mutants had no detectable effect either on signal recognition by mammalian signal recognition particle or on the efficiency of translocation itself.

Amino Acid Sequence↗
PubMed1987

Effects of prolipoprotein signal peptide mutations on secretion of hybrid prolipo-beta-lactamase in Escherichia coli.

Hybrid proteins were constructed by coupling beta-lactamase to the signal sequence (plus nine amino acids) of selected mutant prolipoproteins of Escherichia coli. The mutant prolipoprotein signal peptides contained lesions in two structural domains of the signal peptide, the basic amino-terminal domain and the hydrophobic core domain. We then compared the processing and localization of the mutant prolipo-beta-lactamases to the processing and localization of the comparable mutant prolipoproteins. We show that a mutant signal sequence with an anionic amino terminus exhibits similar limitations in the processing of prolipo-beta-lactamase as previously observed in prolipoprotein. Deletion of four hydrophobic residues from hydrophobic core results in a signal peptide which slowly translocates a fraction of the total mutant hybrid protein synthesized. This signal peptide was previously shown to translocate lipoprotein efficiently. Alteration of this hydrophobic core, which stimulated synthesis of mutant prolipoproteins, does not stimulate synthesis of prolipo-beta-lactamase. Finally mutations that slowed processing of prolipoprotein by affecting the proposed helical structure of the signal peptide had no significant effect on the processing of prolipo-beta-lactamase. These results suggest that the positively charged amino-terminal domain of the signal peptide has a common role in protein secretion regardless of the secretory protein. On the other hand, other domains of the signal peptide exhibit different phenotypes when the secretory protein is changed.

Amino Acid Sequence↗
PubMed1987

Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli.

Subtilisin E, an alkaline serine protease of Bacillus subtilis 168, is first produced as a precursor, pre-pro-subtilisin, which consists of a signal peptide for protein secretion (pre-sequence) and a peptide extension of 77 amino acid residues (pro-sequence) between the signal peptide and mature subtilisin. When the entire coding region for pre-pro-subtilisin E was cloned into an Escherichia coli expression vector, active mature subtilisin E was secreted into the periplasmic space. When the pre-sequence was replaced with the E. coli OmpA signal peptide, active subtilisin E was also produced. When the OmpA signal peptide was directly fused to the mature subtilisin sequence, no protease activity was detected, although this product had the identical primary structure as subtilisin E as a result of cleavage of the OmpA signal peptide and was produced at a level of approximately 10% of total cellular protein. When the OmpA signal peptide was fused to the 15th or 44th amino acid residue from the amino terminus of the pro-sequence, active subtilisin was also not produced. These results indicate that the pro-sequence of pre-pro-subtilisin plays an important role in the formation of enzymatically active subtilisin. It is proposed that the pro-sequence is essential for guiding appropriate folding of the enzymatically active conformation of subtilisin E.

Amino Acid Sequence↗
PubMed1987

The isolation and characterization of RNA coded by the micF gene in Escherichia coli.

A new species of micF RNA, which contains 93 nucleotides (a 4.5S size), was isolated from Escherichia coli. The sequence of the 4.5S micF RNA corresponds to positions G82 through U174 of the micF gene. The 5' terminal end of this smaller micF RNA is triphosphorylated signifying that it is a primary transcript. Its promoter region, which is situated within the greater micF structural gene, has been identified and characterized by lacZ fusion analysis. A 6S micF RNA species, which has a base composition predicted for a transcript from the full length gene has also been detected; however, the 4.5S micF RNA is the predominant species. The work clearly shows by biochemical identification the presence of chromosomally encoded micF RNA.

Bacterial Outer Membrane Proteins↗
PubMed1987

The differential effect on two hybrid proteins of deletion mutations within the hydrophobic region of the Escherichia coli OmpA signal peptide.

Oligonucleotide-directed site-specific mutagenesis was used to systematically shorten the hydrophobic region within the signal peptide of the Escherichia coli outer membrane protein OmpA. DNA encoding the wild type and mutant OmpA signal peptides were then fused in frame to DNA encoding the mature regions of Staphylococcus aureus nuclease A and TEM beta-lactamase. The ability of these signal peptides to direct processing of the resulting hybrid proteins was dependent on both their length and the protein to which they were fused. Deletion of two or more residues progressively slowed processing of pro-OmpA-nuclease. By contrast, pro-OmpA-beta-lactamase was less sensitive to the length of the hydrophobic region than to the nature of the deleted residue(s). Deletion of an Ala residue tended to reduce processing efficiency of pro-OmpA-beta-lactamase, while deletion of an Ile residue, together with the Ala residue, resulted in improvement. The loss of either 3 or 4 residues abolished processing of both hybrids. These data indicate that both the length as well as the identity of residues in the hydrophobic region are important. The relative importance of these two factors depends on the mature region of the protein being secreted.

Amino Acid Sequence↗
PubMed1987

Branched RNA covalently linked to the 5' end of a single-stranded DNA in Stigmatella aurantiaca: structure of msDNA.

Stigmatella aurantiaca is a gliding, gram-negative bacterium that shows a spectacular fruiting body formation upon starvation of nutrient. This bacterium was found to contain approximately 500 copies per cell of a short single-stranded linear DNA (multicopy single-stranded DNA: msDNA). The primary structure of msDNA was determined and found to consist of 162 or 163 deoxyribonucleotides. Its unique chromosomal gene was cloned and sequenced. The msDNA was found to be attached to a branched RNA by its 5' end. Structural analysis of the branched RNA revealed that it consists of a triribonucleotide, 5'A-G-(C or U)3', and that msDNA is branched out from the 2' position of the rG residue forming a 2', 5' phosphodiester linkage with the dC residue at the 5' end of msDNA.

Base Sequence↗
PubMed1987

Biosynthesis and structure of stable branched RNA covalently linked to the 5' end of multicopy single-stranded DNA of Stigmatella aurantiaca.

Stigmatella aurantiaca, a gram-negative bacterium, contains approximately 500 copies per cell of a short single-stranded linear DNA (multicopy single-stranded DNA: msDNA). This DNA is attached to a branched RNA (msdRNA) by its 5' end. The entire sequence of msdRNA was determined and found to consist of 76 bases. The msDNA is linked at the 19th G residue of msdRNA by a 2', 5' phosphodiester linkage. The coding region for msdRNA (msr) is located downstream of the coding region for msDNA (msd). These coding regions exist in opposite orientation with respect to each other and overlap by 8 bases at their 3' ends. Biosynthesis of RNA-linked msDNA was characterized and mechanisms of synthesis are proposed.

Base Sequence↗
PubMed1987

Overproduction of an antisense RNA containing the oop RNA sequence of bacteriophage lambda induces clear plaque formation.

We have constructed an IPTG-inducible plasmid which overexpresses oop RNA sequences in Escherichia coli. Infection of these transformed E. coli cells (SB221/pOOP5) with lambda+ phage produced clear plaques, whereas lambda+ infection of cells transformed with the plasmid vector (SB221/pJDC406) or the plasmid expressing the oop RNA transcript in the other orientation (SB221/pOOP9) gave rise to turbid plaques characteristic of lambda+. Calculations of the percentage of infected cells forming lysogens show a 6-fold decrease in the absence of isopropyl beta-D-thiogalactoside (IPTG) and a 20-fold decrease in the presence of IPTG for SB221/pOOP5 as compared to both SB221/pJDC406 and SB221/pOOP9. We have thus shown that the overexpression of oop RNA favors the lytic mode of lambda development.

Bacteriophage lambda↗
PubMed1987

Use of GC/MS/SIM for rapid determination of plasma levels of o,p'-DDD, o,p'-DDE and o,p'-DDA.

A new method for extraction and quantification of plasma o,p'-DDD (2,2-(2-chlorophenyl,4'-chlorophenyl)1,1-dichloroethane) and its metabolites has been developed. When plasma (0.1 ml) adsorbed to and dried on a filter paper was heated with 5% hydrogen chloride in methanol (1 ml) in a boiling water bath, o,p'-DDD and its metabolites were liberated from the serum protein and were able to be easily extracted with benzene. The recovery rate was raised compared with conventional methods. In addition, this procedure also carried out the simultaneous methylation of o,p'-DDA, one of the metabolites. Mass numbers of 199, 210, 235 and 246 were selected from the mass spectra of o,p'-DDD and its related substances, and they were monitored. Identification was performed on the basis of the retention time and the mass peak intensity ratio. Quantitative determination was performed using the internal standard technique. It was learned that o,p'-DDA is present in the plasma at a concentration about 10 times higher than the levels of o,p'-DDD and o,p'-DDE.

Circadian Rhythm↗
PubMed1987

Precise localization of an overproduced periplasmic protein in Escherichia coli: use of double immuno-gold labelling.

The subcellular localization of beta-lactamase produced by a secretion-cloning vector pIN-III was studied by immunolabelling of frozen thin sections of Escherichia coli. Using double immuno-gold detections and internal reference proteins, it is shown here that beta-lactamase encoded by this vector can be exported and that its overproduction leads to aggregation within the periplasm. This aggregation induces the appearance of electron-dense areas immunolabelled by the antiserum directed against the beta-lactamase at the external side of the cytoplasmic membrane. The overproduced enzyme is also secreted to the medium in vesicles budding from the outer membrane of lpp strains.

Escherichia coli↗
PubMed1987

Distribution of newly synthesized lipoprotein over the outer membrane and the peptidoglycan sacculus of an Escherichia coli lac-lpp strain.

The insertion of newly synthesized lipoprotein molecules into the cell wall of Escherichia coli was studied topographically by immunoelectron microscopy. Lipoprotein was briefly induced with isopropyl-beta-D-thiogalactopyranoside in cells carrying lac-lpp on a low-copy-number plasmid in an E. coli lpp host. Specific antibodies bound to the newly inserted lipoprotein molecules, which were exposed at the cell surface after treatment of the cells with Tris-EDTA, were detected with a protein A-gold probe. The average distribution of the gold particles over the cell surface of noninduced cells was determined for cells induced for 5 and 10 min. Analysis of 250 to 350 cells showed that the distribution of newly synthesized lipoprotein over the cell surface was homogeneous in both cases. The binding of lipoprotein to the peptidoglycan layer was studied by the same technique, and visual inspection again revealed a homogeneous distribution of bound lipoprotein over the entire sacculus surface. It is therefore concluded that free lipoprotein is inserted equally over the entire cell wall of E. coli, while binding to peptidoglycan also occurs over the entire cell surface. The rate of lipoprotein synthesis increased with cell length in nondividing cells, whereas it was constant in cells which had initiated constriction. Analysis of cells having different amounts of lipoprotein in their cell wall revealed that the cell shape depended on the total lipoprotein content of the cell. Cells having no or only a small amount of lipoprotein grew as spheres, whereas cells with increasing numbers of lipoprotein molecules gradually changed their shape to short rods.

Bacterial Outer Membrane Proteins↗
PubMed1987

Expression of the Kirsten ras viral and human proteins in Escherichia coli.

The expression vectors pINIII-A and pINIII (lpp p5) were used to construct plasmids which direct the synthesis in Escherichia coli of the Kirsten ras viral (v-Ki-ras) and human cellular (c-Ki-ras) oncogene products as fusion proteins containing 9 and 10 extra amino acids, respectively, at their N termini. Authenticity of the bacterially produced proteins was determined by immunoprecipitation and immunoblot analyses with ras-specific monoclonal antibodies. After induction with isopropyl-beta-D-thiogalactopyranoside, the viral protein represented approximately 20% of the total cellular protein. The majority of the protein was found in the postsonication low-speed centrifugation pellet. The synthesized viral protein was active in GTP binding, as judged by autophosphorylation and photoaffinity labeling assays.

DNA Restriction Enzymes↗
PubMed1986

Interaction of a transcriptional activator, OmpR, with reciprocally osmoregulated genes, ompF and ompC, of Escherichia coli.

The ompB locus, comprised of the genes ompR and envZ, regulates the expression of the genes ompF and ompC that encode the major porin proteins in the outer membrane of Escherichia coli K-12. OmpR is believed to activate transcription of ompF and ompC in a reciprocal manner depending upon the osmolarity of the culture medium. We were able to purify OmpR to homogeneity and to characterize its interaction with the porin gene promoters. The purified OmpR was shown to bind specifically to promoter fragments of both ompF and ompC. Deoxyribonuclease I footprinting was carried out in order to determine binding sites of OmpR in the promoter regions of the ompF and ompC. OmpR was found to protect the regions -105 to -60 of ompF and -102 to -78 of ompC, respectively. Two consensus sequences were found in these protected sites and are likely to play an important role in OmpR recognition of these promoter regions. The purified OmpR was also shown to be functionally active in transcription in vitro; OmpR activates the expression of ompF and inhibits the transcription of ompC from the most distal of its three tandem promoters, P3.

Bacterial Outer Membrane Proteins↗
PubMed1986

Structural requirement at the cleavage site for efficient processing of the lipoprotein secretory precursor of Escherichia coli.

A phenotypically silent mutation in the signal peptide of the Escherichia coli outer membrane prolipoprotein was combined with other mutations in the mature lipoprotein structure. Under conditions where the individual mutations permit normal lipoprotein secretion, the prolipoprotein with both mutations was unable to be normally modified or processed. These results demonstrate that a given signal peptide is fully functional only if it is structurally compatible with the protein to be secreted. This structural compatibility between the signal peptide and the secretory protein is considered to be dependent on the secondary structure formed at or near the signal peptide cleavage site.

Amino Acid Sequence↗