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C Ueguchi

Publications and source records attributed to C Ueguchi.

48 records · Page 3Linked to original sources
PubMed1993

Function of the Escherichia coli nucleoid protein, H-NS: molecular analysis of a subset of proteins whose expression is enhanced in a hns deletion mutant.

The expression of numerous Escherichia coli cellular proteins was previously demonstrated to be greatly enhanced in a hns deletion background, relative to the levels in wild-type cells. In this study, a subset of such proteins, expression of which is affected by H-NS, was partially purified, and the genes coding for some of the proteins were identified and characterized. Two of the proteins thus characterized, 19K and 17K, were found to be encoded by previously predicted genes that are located adjacent to, and downstream of, the trpABCDE operon (27.6 min on the E. coli genetic map). The genes coding for the other two proteins, 10K-L and 10K-S, are located at 77.5 min on the genetic map. Their nucleotide sequences were determined and revealed that they may constitute an operon. To characterize the putative promoters for these genes, a set of promoter-lacZ transcriptional fusion genes was constructed on the E. coli chromosome. The results of such promoter-probe analyses indicated that H-NS represses the expression of these genes at the transcriptional level. Furthermore, H-NS appeared to exhibit relatively strong affinity for the putative promoter sequences in vitro. These results are compatible with the hypothesis that H-NS functions as a transcriptional repressor.

Amino Acid Sequence↗
PubMed1992

Insertional disruption of the nusB (ssyB) gene leads to cold-sensitive growth of Escherichia coli and suppression of the secY24 mutation.

The Escherichia coli gene ssyB was cloned and sequenced. The ssyB63 (Cs) mutation is an insertion mutation in nusB, while the nusB5 (Cs) mutation suppresses secY24, indicating that inactivation of nusB causes cold-sensitive cell growth as well as phenotypic suppression of secY24. The correct map position of nusB is 9.5 min rather than 11 min as previously assigned. It is located at the distal end of an operon that contains a gene showing significant homology with a Bacillus subtilis gene involved in riboflavin biosynthesis.

Amino Acid Sequence↗
PubMed1992

Multicopy suppression: an approach to understanding intracellular functioning of the protein export system.

Escherichia coli genes were cloned onto a multicopy plasmid and selected by the ability to restore growth and protein export defects caused by a temperature-sensitive secY or secA mutation. When secA51 was used as the primary mutation, only clones carrying groE, which specifies the chaperonin class of heat shock protein, were obtained. Selection using secY24 yielded three major classes of genes. The first class encodes another heat shock protein, HtpG; the most frequently obtained second class encodes a neutral histonelike protein, H-NS; and the third class, msyB, encodes a 124-residue protein of which 38 residues are acidic amino acids. Possible mechanisms of suppression as well as the significance and limitations of the multicopy suppression approach are discussed.

Amino Acid Sequence↗
PubMed1991

Yeast gene which suppresses the defect in protein export of a secY mutant of E. coli.

To find factors participating in protein translocation in yeast, we screened a yeast genomic library for genes which, when introduced into Escherichia coli, suppressed secY24, a temperature sensitive mutation of an essential integral membrane protein (SecY) required for protein export. We isolated and characterized a gene (YSY6) which improved the translocation of the OmpA protein in mutant strain IQ85(secY24). It could also suppress another mutant [rplO215(Am)], in which the level of expression of the SecY protein is decreased at high-temperature. The YSY6 gene encodes a small amphiphilic peptide consisting of 65 amino acids, which can be expressed in E. coli cells.

Amino Acid Sequence↗
PubMed1990

Escherichia coli sec mutants accumulate a processed immature form of maltose-binding protein (MBP), a late-phase intermediate in MBP export.

Protein translocation across the Escherichia coli cytoplasmic membrane may consist of several temporally or topographically distinct steps. Although early events in the translocation pathway have been characterized to some extent, the mechanisms responsible for the trans-bilayer movement of a polypeptide are only poorly understood. This article reports on our attempts to dissect the translocation pathway in vivo. A processed form of maltose-binding protein (MBP) was detected in the spheroplasts of secY and secA temperature-sensitive mutant cells that had been pulse-labeled at the permissive temperature (30 degrees C). This species of molecule was found to have an electrophoretic mobility identical to that of the mature MBP, but a considerable fraction of it was inaccessible to externally added protease. It had not attained the protease-resistant conformation characteristically observed for the exported mature protein. The radioactivity associated with this species decreased during chase and was presumably converted into the exported mature form, a process that required energy, probably the proton motive force, as demonstrated by its inhibition by an energy uncoupler. The spheroplast-associated processed form was more predominantly observed in the presence of a low concentration of chloramphenicol. A similar intermediate was also detected for beta-lactamase in wild-type cells. These results suggest that in a late phase of translocation, the bulk of the polypeptide chain can move through the membrane in the absence of the covalently attached leader peptide, and the secA-secY gene products are somehow involved in this process. We termed the processed intermediates processed immature forms.

ATP-Binding Cassette Transporters↗
PubMed1990

Characterization of cold-sensitive secY mutants of Escherichia coli.

Mutations which cause poor growth at a low temperature, which affect aspects of protein secretion, and which map in or around secY (prlA) were characterized. The prlA1012 mutant, previously shown to suppress a secA mutation, proved to have a wild-type secY gene, indicating that this mutation cannot be taken as genetic evidence for the secA-secY interaction. Two cold-sensitive mutants, the secY39 and secY40 mutants, which had been selected by their ability to enhance secA expression, contained single-amino-acid alterations in the same cytoplasmic domain of the SecY protein. Protein export in vivo was partially slowed down by the secY39 mutation at 37 to 39 degrees C, and the retardation was immediately and strikingly enhanced upon exposure to nonpermissive temperatures (15 to 23 degrees C). The rate of posttranslational translocation of the precursor to the OmpA protein (pro-OmpA protein) into wild-type membrane vesicles in vitro was only slightly affected by reaction temperatures ranging from 37 to 15 degrees C, and about 65% of OmpA was eventually sequestered at both temperatures. Membrane vesicles from the secY39 mutant were much less active in supporting pro-OmpA translocation even at 37 degrees C, at which about 20% sequestration was attained. At 15 degrees C, the activity of the mutant membrane decreased further. The rapid temperature response in vivo and the impaired in vitro translocation activity at low temperatures with the secY39 mutant support the notion that SecY, a membrane-embedded secretion factor, participates in protein translocation across the bacterial cytoplasmic membrane.

Bacterial Outer Membrane Proteins↗
PubMed1989

Effects of mutations in heat-shock genes groES and groEL on protein export in Escherichia coli.

Escherichia coli heat-shock proteins GroES and GroEL are essential cytoplasmic proteins, which have been termed 'chaperonins' because of their ability to assist protein assembly of bacteriophage capsids and multimeric enzymes of foreign origin. In this report we show that temperature-sensitive mutations in groES and groEL genes cause defective export of the plasmid-encoded beta-lactamase (Bla) in vivo. Since efficient translocation of proteins across biological membranes is thought to be supported by cytoplasmic factors that protect presecretory molecules from being misfolded, these results suggest that both GroES and GroEL proteins possess a chaperone function by which they facilitate export of Bla. The translocation of other secretory proteins, however, appears to depend minimally on GroE, suggesting that GroE interacts only with a specific class of secreted proteins.

Antigens, Bacterial↗
PubMed1989

The secY-rpmJ region of the spc ribosomal protein operon in Escherichia coli: structural alterations affecting secY expression.

A unique feature of the spc ribosomal protein operon is that its region distal to the promoter contains a gene (secY) for an integral membrane protein, followed by an open reading frame termed X which has recently been proposed to encode a new ribosomal protein (protein B). We now show that the open reading frame X indeed directs the synthesis of a protein with electrophoretic mobilities similar to the B protein, and this supports the proposal that X may be more appropriately called rpmJ. Insertion of a plasmid sequence into the secY-rpmJ boundary of the chromosome caused a reduced expression of secY probably by destabilizing the secY part of the message. The results of complementation experiments suggested that a normal level of expression of rpmJ is not required for growth or protein secretion.

Bacterial Proteins↗