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F Omura

Publications and source records attributed to F Omura.

At least 19 recordsLinked to original sources
PubMed2001

The N-terminal domain of the yeast permease Bap2p plays a role in its degradation.

The amino acid permease Bap2p in Saccharomyces cerevisiae mediates a major part of the uptake of leucine, isoleucine, and valine from media containing a preferred nitrogen source. Although the transcriptional controls of BAP2 have been well studied, the posttranslational down-regulation mechanisms for Bap2p have not been established. Here we show that Bap2p is subject to a starvation-induced degradation upon rapamycin treatment or cultivation with proline as the sole nitrogen source. The starvation-induced degradation of Bap2p was dependent on the cellular functions of ubiquitination and endocytosis. Down-regulation of the permease required the most probable ubiquitination sites, the lysine residues situated in the N-terminal 49 residues, as well as the C-terminal domain. Furthermore, when the N-terminal domain of Bap2p was fused to the general amino acid permease Gap1p, the resultant chimeric permease became susceptible to the starvation-induced degradation, indicating that the Bap2p N-terminus contains a determinant responsive to the starvation signals.

Amino Acid Sequence↗
PubMed2001

The basal turnover of yeast branched-chain amino acid permease Bap2p requires its C-terminal tail.

The branched-chain amino acid permease Bap2p is a transport system for leucine, isoleucine, and valine in Saccharomyces cerevisiae, and its synthesis is regulated transcriptionally. However, the downregulation mechanisms of Bap2p have not been established. Here we demonstrate that the C-terminal region of Bap2p plays a pivotal role in its basal turnover. Truncation of the C-terminal 29 residues caused the stabilization and accumulation in the plasma membrane of Bap2p. Furthermore, when the Bap2p C-terminal region was fused to green fluorescent protein, the fusion protein localized to the plasma membrane, suggesting the existence of a possible degradation-related acceptor site for the C-terminal tail of Bap2p.

Amino Acid Transport Systems↗
PubMed2001

Isolation and characterization of a gene specific to lager brewing yeast that encodes a branched-chain amino acid permease.

We found two types of branched-chain amino acid permease gene (BAP2) in the lager brewing yeast Saccharomyces pastorianus BH-225 and cloned one type of BAP2 gene (Lg-BAP2), which is identical to that of Saccharomyces bayanus (by-BAP2-1). The other BAP2 gene of the lager brewing yeast (cer-BAP2) is very similar to the Saccharomyces cerevisiae BAP2 gene. This result substantiates the notion that lager brewing yeast is a hybrid of S. cerevisiae and S. bayanus. The amino acid sequence homology between S. cerevisiae Bap2p and Lg-Bap2p was 88%. The transcription of Lg-BAP2 was not induced by the addition of leucine to the growth medium, while that of cer-BAP2 was induced. The transcription of Lg-BAP2 was repressed by the presence of ethanol and weak organic acid, while that of cer-BAP2 was not affected by these compounds. Furthermore, Northern analysis during beer fermentation revealed that the transcription of Lg-BAP2 was repressed at the beginning of the fermentation, while cer-BAP2 was highly expressed throughout the fermentation. These results suggest that the transcription of Lg-BAP2 is regulated differently from that of cer-BAP2 in lager brewing yeasts.

Amino Acid Sequence↗
PubMed1998

[Proliferative potential of the ear drum in the process of gerbiline cholesteatoma formation].

Mongolian gerbil frequently develop spontaneous cholesteatoma. As we reported previously, in the process of gerbiline cholesteatoma formation, effusions inside the pars flaccida are always found in the ears during the early stage, and epidermal growth factor (EGF) is also localized in the pars flaccida, especially in the mucous layer. In this study, to clarify the process of gerbiline cholesteatoma formation, we studied 22 gerbiline temporal bones by using a monoclonal antibody against bromodeoxyuridine (BrdU). BrdU-labeled cells demonstrate a proliferative potential. We also used a carbon dye method to label the micro-vase in 14 gerbiline temporal bones. Cells showing BrdU uptake were more abundant, as demonstrated immunohistochemically, in the pars flaccida of ears with early cholesteatomas than in the pars flaccida of normal ears (p < 0.01). The pars flaccida of ears with early cholesteatomas showed hypertrophy of both epithelial layers, and hyperkeratosis of the epidermal layer. BrdU-labeled cells in the pars flaccida were more localized in the mucous layers than in the epidermal layers. In contrast, in ears with cholesteatomas, BrdU-labeled cells were less abudant than in ears with early cholesteatomas. In addition, BrdU-labeled cells in the pars tensa and external auditory epidermal layers were not increased in ears with any stage of cholesteatoma formation. We used a carbon dye method to detect the micro-vasa in the intermediate layer of the ear drum. Carbon-dye-labeled vasa were more numerous in the pars flaccida with early cholesteatomas than in the pars flaccida of normal ears or ears with cholesteatomas. It is highly suspected that angiogenesis was stimulated in the pars flaccida with early cholesteatomas, because stimulation of angiogenesis by EGF has been reported. The above findings suggest that the mucous layer of the pars flaccida has the greatest proliferative potential in the process of cholesteatoma formation. Angiogenesis in the pars flaccida appears to be a reaction to proliferative changes in the mucous and epidermal layers. These changes are probably stimulated by effusion inside the pars flaccida.

Animals↗
PubMed1996

Single point mutations in Met4p impair the transcriptional repression of MET genes in Saccharomyces cerevisiae.

Transcription of MET genes in Saccharomyces cerevisiae depends on a transcriptional activator, the MET4 gene product (Met4p). Using in vitro mutagenesis, we isolated two mutant MET4 alleles encoding [Pro215]Met4p and [Ser156]Met4p. These mutations impeded Met4p's responsiveness to methionine in the media, and yeast cells carrying mutant alleles exhibited enhanced transcription of MET genes under repressing conditions. The enhanced transcription was dependent on the CBF1 gene, but did not compete with an excess of wild-type Met4p, suggesting that some changes in the affinity of Met4p to other factors might be involved in S-adenosylmethionine-mediated transcriptional regulation.

Alleles↗
PubMed1995

A possible role of ER-60 protease in the degradation of misfolded proteins in the endoplasmic reticulum.

Wild-type human lysozyme (hLZM) is secreted when expressed in mouse L cells, whereas misfolded mutant hLZMs are retained and eventually degraded in a pre-Golgi compartment (Omura, F., Otsu, M., Yoshimori, T., Tashiro, Y., and Kikuchi, M. (1992) Eur. J. Biochem. 210, 591-599). These misfolded mutant hLZMs are associated with protein disulfide isomerase (Otsu, M., Omura, F., Yoshimori, T., and Kikuchi, M. (1994) J. Biol. Chem. 269, 6874-6877). From the observation that this degradation is sensitive to cysteine protease inhibitors, such as N-acetyl-leucyl-leucyl-norleucinal and N-acetyl-leucyl-leucyl-methioninal, but not to the serine protease inhibitors, 1-chloro-3-tosylamido-7-amino-2-heptanone and (p-amidinophenyl)methanesulfonyl fluoride, it was suggested that some cysteine proteases are likely responsible for the degradation of abnormal proteins in the endoplasmic reticulum (ER). ER-60 protease (ER-60), an ER resident protein with cysteine protease activity (Urade, R., Nasu, M., Moriyama, T., Wada, K., and Kito, M. (1992) J. Biol. Chem. 267, 15152-15159), was found to associate with misfolded hLZMs, but not with the wild-type protein, in mouse L cells. Furthermore, denatured hLZM is degraded by ER-60 in vitro, whereas native hLZM is not. These results suggest that ER-60 could be a component of the proteolytic machinery for the degradation of misfolded mutant hLZMs in the ER.

Animals↗
PubMed1995

The role of middle ear effusions and epidermal growth factor in cholesteatoma formation in the gerbilline temporal bone.

To study the process of aural cholesteatoma formation, we used gerbilline temporal bones to examine histologically the early stages of spontaneous cholesteatomas associated with experimentally induced otitis media with effusion (OME) following electric cauterizations of the eustachian tube. Epidermal growth factor (EGF) was then localized immunohistochemically in the pars flaccida of normal ears and the forming spontaneous cholesteatomas. Findings in the ears with the early spontaneous cholesteatomas were effusion inside the pars flaccida and hypertrophy and hyperkeratosis of the pars flaccida. Findings in the ears with experimental OME involved an effusion in the whole middle ear cavity as well as hypertrophy and hyperkeratosis in both the pars flaccida and pars tensa. The incidence of ear drum changes was higher in the experimental OME group than in control animals without cauterization. EGF was localized in the mucous layer of normal drums, the mucous layer and lamina propria of drums with hypertrophy alone, and all layers in drums with hypertrophy and hyperkeratosis. EGF was especially positive in the cytoplasms of transformed cuboidal cells. These findings suggest that EGF within the transformed mucous layer may play an important role as a biochemical factor in developing cholesteatomas.

Animals↗
PubMed1994

Protein disulfide isomerase associates with misfolded human lysozyme in vivo.

Wild-type human lysozyme (hLZM) is quantitatively secreted into the media when expressed in mouse fibroblast cells, but some misfolded hLZMs are retained and rapidly degraded in a pre-Golgi compartment (Omura, F., Otsu, M., Yoshimori, T., Tashiro, Y., and Kikuchi, M. (1992) Eur. J. Biochem. 210, 591-599). To detect the association with misfolded hLZMs of cellular proteins involved in their folding, retention, and pre-Golgi degradation, a co-precipitation experiment was carried out using anti-hLZM antibody and metabolically labeled cell lysates, which were treated with a membrane-permeable cross-linking reagent. Here we report that protein disulfide isomerase associated in vivo with misfolded hLZMs, but not with the wild-type protein, and discuss the possible role of protein disulfide isomerase in the quality control of newly synthesized proteins in the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1994

Structure and expression of a gene coding for thermostable alpha-glucosidase with a broad substrate specificity from Bacillus sp. SAM1606.

We cloned an alpha-glucosidase gene from thermophilic Bacillus sp. SAM1606 to overexpress it in Escherichia coli transformants. Deletion of the 5'-noncoding region as well as expression of the alpha-glucosidase gene under the control of the icp promotor of the insecticidal crystal protein gene from Bacillus thuringiensis subsp. sotto enhanced the enzyme productivity to 23.5 U/ml, which was 12,000-fold higher than that obtained by the strain SAM1606. The open reading frame corresponding to the alpha-glucosidase encoded 587 amino acid residues including a residue coded by the initiation codon TTG, and the molecular mass of the alpha-glucosidase from N-terminal serine was calculated to be 68,886 Da. Sequence analysis revealed that the SAM1606 alpha-glucosidase belonged to the alpha-amylase family. The SAM1606 alpha-glucosidase showed extremely high sequence identity (62-65%) to the Bacillus cereus and Bacillus thermoglucosidasius oligo-1,6-glucosidases, which were 72% identical to each other. Sequence identity in the suggested active site regions were essentially the same (80-82%) among these three enzymes. However, the substrate specificity of the SAM1606 alpha-glucosidase was significantly different from those of the oligo-1,6-glucosidases. The thermostability of these three alpha-glucosidases could be correlated with the increase in the number of proline residues, whose occurrence was predicted at beta turns and coils in the enzymes.

Amino Acid Sequence↗
PubMed1993

N200 component of event-related potentials in depression.

Event-related potentials (ERPs) recorded during a two-tone discrimination (oddball) task were examined in 36 drug-free depressed patients and 36 control subjects. At remission, the ERPs of 12 of the depressed patients were reexamined. In the depressed patients, although a group difference was not detected in the peak latency and amplitude of N200 to rare stimuli, the mean amplitude for the N200 latency range in the difference waves was smaller than in the control subjects. Mismatch negativity (N2a), which was elicited by rare stimuli, was reduced in amplitude; but N2b may have been evoked to frequent stimuli more in the patients than in the control subjects. Depressed subjects may have a deviance in the fully automatic cerebral mismatch process that is assumed to be related to mismatch negativity and provoke the controlled mismatch detection process (presumed to be associated with N2b) even to nontarget frequent stimuli. These findings were observed during remission; however, there was a tendency for the N2b amplitude to decrease and recover toward the level of the control subjects.

Adolescent↗
PubMed1992

Non-lysosomal degradation of misfolded human lysozymes with and without an asparagine-linked glycosylation site.

Human lysozyme is a monomeric secretory protein composed of 130 amino acid residues, with four intramolecular disulfide bonds and no oligosaccharides. In this study, a mutant protein, [Ala128] lysozyme, which cannot fold because it lacks a disulfide bond, Cys6-Cys128, was expressed in mouse fibroblasts and was found to be mostly degraded in the cells, whereas the control wild-type lysozyme was quantitatively secreted into the media. The degradation of [Ala128]lysozyme was independent of the transport from the endoplasmic reticulum to the Golgi apparatus. The degradation was greatly inhibited by incubation of cells at 15 degrees C, but was minimally affected by treatment of cells with the lysosomotropic agent, chloroquine, implying a non-lysosomal process. Additional mutations (Gly48-->Ser or Met29-->Thr) were created to make asparagine-linked (N-linked) glycosylation site in the [Ala128]lysozyme, and the resultant double mutants, [Ser48, Ala128]lysozyme and [Thr29, Ala128]lysozyme, were analyzed with respect to their intracellular degradation. These mutant proteins were susceptible to N-linked glycosylation, and were degraded in a similar manner to that of [Ala128] lysozyme, except that the onset of degradation of [Ser48, Ala128]lysozyme and [Thr29, Ala128] lysozyme, but not of [Ala128]lysozyme, was preceded by a lag period of up to 60 min. Furthermore, the degradative double mutants, [Ser48, Ala128]lysozyme and [Thr29, Ala128]lysozyme, were glycosylated post-translationally as well as co-translationally. These observations suggest that there is some interaction between the mechanisms of glycosylation and degradation.

Animals↗
PubMed1992

Accelerated secretion of human lysozyme with a disulfide bond mutation.

The mutant human lysozyme, [Ala77, Ala95]lysozyme, in which the disulfide bond Cys77-Cys95 is eliminated, is known to exhibit increased secretion in yeast, compared to wild-type human lysozyme [Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M. & Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967]. To investigate this phenomenon, mammalian cells were used to analyze the secretion kinetics of [Ala77, Ala95]lysozyme and wild-type human lysozyme. The secretion rate of [Ala77, Ala95]lysozyme during the 150-min chase period was significantly accelerated [half-life (t1/2) = 29 min] compared to that of wild-type human lysozyme (t1/2 = 83 min), when expressed at the same levels within the cells. In contrast, after the 150-min chase, the rates of disappearance of both wild-type and mutant human lysozymes within the cells were similar, and considerably slower (t1/2 = 220 min), respectively. The remaining intracellular wild-type human lysozyme was localized mainly in the endoplasmic reticulum, whereas accelerated transport of the [Ala77, Ala95]lysozyme mutant protein from the endoplasmic reticulum to the Golgi apparatus was observed. Also in yeast cells, similar secretion kinetics and the differences in t1/2 for wild-type and mutant human lysozymes during the early chase period were observed. The two-phase kinetics of disappearance of intracellular human lysozymes suggest that only a proportion of the proteins becomes secretion competent soon after synthesis and is completely secreted during the early chase period, whereas others enter the distinct, slow pathways of intracellular transport and/or degradation. Increased secretion of [Ala77, Ala95]lysozyme is possibly due to enhanced competence for secretion acquired in the endoplasmic reticulum at the early stage of transport events, which is closely connected with the removal of a disulfide bond.

Adenocarcinoma↗
PubMed1991

Behavior of cysteine mutants of human lysozyme in de novo synthesis and in vivo secretion.

To investigate the mechanism of disulfide-bond-coupled de novo folding of human lysozyme, we have constructed 23 mutant enzymes in which cysteine residue(s) were replaced by alanine(s). The mutant genes were translated in vitro in a system composed of rabbit reticulocyte lysate, canine pancreatic microsomal vesicles and oxidized glutathione. This system allows the formation of intramolecular disulfide bonds in translation products translocated into the microsomal lumen. The mobilities of the translation products were analyzed by SDS/PAGE in nonreducing conditions. Some mutant lysozymes were found to form a compact conformation with native-like mobility in the presence of SDS. The de novo formation of the SDS-resistant compact conformation of each mutant correlated well with its efficiency of secretion by Saccharomyces cerevisiae. Our results suggest that the de novo synthesized products reflect the conformational states in vivo to some extent, and that the formation of SDS-resistant compact conformation can be regarded as a necessary condition for allowing lysozyme to be secreted. In addition, the analysis of a mutant C116A (Cys116----Ala) under different oxidative conditions suggests two distinct pathways for the disulfide-bond-coupled formation of the compact conformation.

Animals↗
PubMed1991

Evidence for intramolecular disulfide bond shuffling in the folding of mutant human lysozyme.

Our previous results using the Saccharomyces cerevisiae secretion system suggest that intramolecular exchange of disulfide bonds occurs in the folding pathway of human lysozyme in vivo (Taniyama, Y., Yamamoto, Y., Kuroki, R., and Kikuchi, M. (1990) J. Biol. Chem. 265, 7570-7575). Here we report on the results of introducing an artificial disulfide bond in mutants with 2 cysteine residues substituting for Ala83 and Asp91. The mutant (C83/91) protein was not detected in the culture medium of the yeast, probably because of incorrect folding. Thereupon, 2 cysteine residues Cys77 and Cys95 were replaced with Ala in the mutant C83/91, because a native disulfide bond Cys77-Cys95 was found not necessary for correct folding in vivo (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). The resultant mutant (AC83/91) was secreted as two proteins (AC83/91-a and AC83/91-b) with different specific activities. Amino acid and peptide mapping analyses showed that two glutathiones appeared to be attached to the thiol groups of the cysteine residues introduced into AC83/91-a and that four disulfide bonds including an artificial disulfide bond existed in the AC83/91-b molecule. The presence of cysteine residues modified with glutathione may indicate that the non-native disulfide bond Cys83-Cys91 is not so easily formed as a native disulfide bond. These results suggest that the introduction of Cys83 and Cys91 may act to suppress the process of native disulfide bond formation through disulfide bond interchange in the folding of human lysozyme.

Amino Acid Sequence↗
PubMed1991

Abnormalities in event-related potentials, N100, P200, P300 and slow wave in schizophrenia.

Event-related potentials were recorded in 54 schizophrenics and 88 age-matched controls during a two-tone discrimination (odd ball) task. All the subjects were free from medication. In the schizophrenics, the mean amplitudes of the N100, P300 and Slow Wave latency ranges were decreased, and the amplitude of the P200 latency range was greater than that for the controls. These reductions and the increase were found both for the ERPs elicited by rare target stimuli and for those elicited by frequent nontarget stimuli. The peak latency of N200 to rare stimuli was more prolonged in the schizophrenics than in the controls. This finding confirms the prolongation of N200 latency that Brecher et al. (1987) found for a different visual stimuli task. Neither the N100 nor P300 latency differed between the two groups.

Adult↗
PubMed1989

The histidine residue of codon 715 is essential for function of elongation factor 2.

Several mutant cDNAs of elongation factor 2 (EF-2) were constructed by site-directed mutagenesis and their products expressed in mouse cells were investigated. Amino acid substitution for the histidine residue of codon 715, which is modified post-translationally to diphthamide, resulted in non-functional EF-2 and this substitution did not render EF-2 resistant to Pseudomonas aeruginosa exotoxin A, which inactivates EF-2 transferring ADP-ribose to the diphthamide residue. These non-functional EF-2s with replacements of the histidine-715 residue showed various extents of inhibition of protein synthesis by competing with functional EF-2 in vivo. These results suggest that histidine-715 is essential for the translocase activity of EF-2 and that the region around diphthamide functions in recognition of, and/or binding to ribosomes. Substitution of proline for the alanine-713 residue and substitution of glutamine for the glycine-717 residue converted EF-2 to partially toxin-resistant forms. Two-dimensional gel analysis with fragment A of diphtheria toxin of these toxin-resistant EF-2s revealed that their ADP-ribosylations by toxin were much less than that of wild-type EF-2.

Adenosine Diphosphate Ribose↗