Yoshida Biological Laboratory

Kyoto, Japan

Yoshida Biological Laboratory

Kyoto, Japan
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Ueta M.,Yoshida Biological Laboratory | Wada C.,Yoshida Biological Laboratory | Wada C.,Japan Science and Technology Agency | Bessho Y.,RIKEN | And 4 more authors.
Genes to Cells | Year: 2017

Ribosomes routinely prepared from Escherichia coli strain K12 contain intact (70 amino acids) and short (62 amino acids) forms of ribosomal protein L31. By contrast, ribosomes prepared from ompT mutant cells, which lack protease 7, contain only intact L31, suggesting that L31 is cleaved by protease 7 during ribosome preparation. We compared ribosomal subunit association in wild-type and ompT− strains. In sucrose density gradient centrifugation under low Mg2+, 70S content was very high in ompT− ribosomes, but decreased in the wild-type ribosomes containing short L31. In addition, ribosomes lacking L31 failed to associate ribosomal subunits in low Mg2+. Therefore, intact L31 is required for subunit association, and the eight C-terminal amino acids contribute to the association function. In vitro translation was assayed using three different systems. Translational activities of ribosomes lacking L31 were 40% lower than those of ompT− ribosomes with one copy of intact L31, indicating that L31 is involved in translation. Moreover, in the stationary phase, L31 was necessary for 100S formation. The strain lacking L31 grew very slowly. A structural analysis predicted that the L31 protein spans the 30S and 50S subunits, consistent with the functions of L31 in 70S association, 100S formation, and translation. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd


Saishin N.,Kobe College | Ueta M.,Yoshida Biological Laboratory | Wada A.,Yoshida Biological Laboratory | Yamamoto I.,Kobe Women's University
Journal of Biological Macromolecules | Year: 2016

Two α-glucosidases, which hydrolyzed 4-nitrophenyl (4NP)-α-D-glucopyranoside, were purified and characterized from Bifidobacterium longum subsp. longum JCM 7052 grown on gum arabic. These enzymes, named BlAglA1 and BlAglA2, had apparent molecular masses of 72.0 and 72.8 kDa estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and 70.1 and 92.7 kDa by PAGE without SDS, respectively. The amino acid sequences analyzed by MALDI-TOF-MS were found in the genomes of several strains of B. longum subsp. longum. BlAglA1 and BlAglA2 showed hydrolyzing activities toward isomaltose, trehalose, palatinose, isomaltotriose and panose, but did not toward sucrose, maltose, amylose, amylopectin, and oyster glycogen, indicating that each enzyme belongs to oligo-1,6-glucosidase (EC 3.2.1.10). Both enzymes showed optimal activities with 4NP-α-D-glucopyranoside at pH 5.5-6.0 and at 45-50°C. Values of Km and Vmax for 4NP-α-D-glucopyranoside, trehalose, palatinose, panose, isomaltose, and isomaltotriose were also determined. The two oligo-1,6-glucosidases were shown to have transglycosylation activities to synthesize oligosaccharides from trehalose, palatinose, isomaltose, panose, and isomaltotriose to their selves. Both enzymes also catalyzed transglucosylation from palati-nose to ethanol, 1-propanol, 2-propanol, 1-butanol, and 1-hexanol.


Kimura K.,Osaka Medical College | Wada A.,Osaka Medical College | Wada A.,Yoshida Biological Laboratory | Ueta M.,Osaka Medical College | And 10 more authors.
International Journal of Oncology | Year: 2010

Many auxiliary functions of ribosomal proteins (r-proteins) have received considerable attention in recent years. However, human r-proteins have hardly been examined by proteomic analysis. In this study, we isolated ribosomal particles and subsequently compared the proteome of r-proteins between the DLD-1 human colon cancer cell line and its 5-fluorouracil (5-FU)-resistant sub-line, DLD-1/5-FU, using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis, which has a superior ability to separate basic proteins, and we discuss the role of r-proteins in 5-FU resistance. Densitometric analysis was performed to quantify modulated proteins, and protein spots showing significant changes were identified by employing matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Three basic proteins (L15, L37 and prohibitin) which were significantly modulated between DLD-1 and DLD-1/5-FU were identified. Two proteins, L15 and L37, showed down-regulated expression in DLD-1/5-FU in comparison to DLD-1. Prohibitin, which is not an r-protein and is known to be localized in the mitochondria, showed up-regulated expression in DLD-1/5-FU. These 3 proteins may be related to 5-FU resistance.


Kato T.,Osaka University | Kato T.,International Cooperative Research Project | Yoshida H.,Osaka Medical College | Miyata T.,Osaka University | And 4 more authors.
Structure | Year: 2010

In the stationary growth phase of bacteria, protein biosynthesis on ribosomes is suppressed, and the ribosomes are preserved in the cell by the formation of the 100S ribosome. The 100S ribosome is a dimer of the 70S ribosome and is formed by the binding of the ribosome modulation factor and the hibernation promoting factor. However, the binding mode between the two 70S ribosomes and the mechanism of complex formation are still poorly understood. Here, we report the structure of the 100S ribosome by electron cryomicroscopy and single-particle image analysis. The 100S ribosome purified from the cell in the stationary growth phase is composed of two transfer RNA-free 70S ribosomes, has two-fold symmetry, and is formed through interactions between their 30S subunits, where interactions between small subunit proteins, S2, S3 and S5, appear to be critical for the dimerization. © 2010 Elsevier Ltd.


Yoshida H.,Osaka Medical College | Maki Y.,Osaka Medical College | Furuike S.,Osaka Medical College | Sakai A.,Osaka Medical College | And 2 more authors.
Journal of Bacteriology | Year: 2012

Here, we provide evidence that YqjD, a hypothetical protein of Escherichia coli, is an inner membrane and ribosome binding protein. This protein is expressed during the stationary growth phase, and expression is regulated by stress response sigma factor RpoS. YqjD possesses a transmembrane motif in the C-terminal region and associates with 70S and 100S ribosomes at the N-terminal region. Interestingly, E. coli possesses two paralogous proteins of YqjD, ElaB and YgaM, which are expressed and bind to ribosomes in a similar manner to YqjD. Overexpression of YqjD leads to inhibition of cell growth. It has been suggested that YqjD loses ribosomal activity and localizes ribosomes to the membrane during the stationary phase. © 2012, American Society for Microbiology.


Ueta M.,Yoshida Biological Laboratory | Wada C.,Yoshida Biological Laboratory | Daifuku T.,Kyoto University | Sako Y.,Kyoto University | And 9 more authors.
Genes to Cells | Year: 2013

In bacteria, 70S ribosomes (consisting of 30S and 50S subunits) dimerize to form 100S ribosomes, which were first discovered in Escherichia coli. Ribosome modulation factor (RMF) and hibernation promoting factor (HPF) mediate this dimerization in stationary phase. The 100S ribosome is translationally inactive, but it dissociates into two translationally active 70S ribosomes after transfer from starvation to fresh medium. Therefore, the 100S ribosome is called the 'hibernating ribosome'. The gene encoding RMF is found widely throughout the Gammaproteobacteria class, but is not present in any other bacteria. In this study, 100S ribosome formation in six species of Gammaproteobacteria and eight species belonging to other bacterial classes was compared. There were several marked differences between the two groups: (i) Formation of 100S ribosomes was mediated by RMF and short HPF in Gammaproteobacteria species, similar to E. coli, whereas it was mediated only by long HPF in the other bacterial species; (ii) RMF/short HPF-mediated 100S ribosome formation occurred specifically in stationary phase, whereas long HPF-mediated 100S ribosome formation occurred in all growth phases; and (iii) 100S ribosomes formed by long HPF were much more stable than those formed by RMF and short HPF. © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.


Doniwa Y.,University of Tokyo | Ueda M.,University of Tokyo | Ueda M.,Kyoto University | Ueta M.,Yoshida Biological Laboratory | And 3 more authors.
Gene | Year: 2010

C-to-U RNA editing (i.e., alteration of a C in the genomic sequence to U in the transcript) has been confirmed widely in angiosperm organellar genomes. During the C-to-U RNA editing event, incomplete edited transcripts have been observed at many sites in the steady-state mRNA population (partial editing). Here, by using coexpression analysis and the surveillance of whole editing status on the mitochondrial genome, we have revealed that a pentatricopeptide repeat (PPR) protein classified into the P subfamily (PPR596) has site-specific influence on the efficiency of C-to-U RNA editing events at partial editing sites on the Arabidopsis thaliana mitochondrial genome. Previous works have revealed that PPR proteins classified into the PLS subfamily containing the E or E and DYW motif are involved in RNA editing as trans-factors; they are believed to recruit deaminase at editing sites. In contrast with the mutant analyses of PLS-subfamily PPR proteins, the editing efficiency at rps3eU1344SS was revealed to be significantly increased in ppr596 mutants. Our study implies P-subfamily PPR protein is involved in the control of the degree of partial editing. © 2010 Elsevier B.V.


Yoshida H.,Osaka Medical College | Wada A.,Yoshida Biological Laboratory
Wiley Interdisciplinary Reviews: RNA | Year: 2014

One of the most important cellular events in all organisms is protein synthesis (translation), which is catalyzed by ribosomes. The regulation of translational activity is dependent on the environmental situation of the cell. A decrease in overall translation under stress conditions is mainly accompanied by the formation of functionally inactive 100S ribosomes in bacteria. The 100S ribosome is a dimer of two 70S ribosomes that is formed through interactions between their 30S subunits. Two mechanisms of 100S ribosome formation are known: one involving ribosome modulation factor (RMF) and short hibernation promoting factor (HPF) in a part of Gammaproteobacteria including Escherichia coli, and the other involving only long HPF in the majority of bacteria. The expression of RMF is regulated by ppGpp and cyclic AMP-cAMP receptor protein (cAMP-CRP) induced by amino acid starvation and glucose depletion, respectively. When stress conditions are removed, the 100S ribosome immediately dissociates into the active 70S ribosomes by releasing RMF. The stage in the ribosome cycle at which the ribosome loses translational activity is referred to as 'Hibernation'. The lifetime of cells that cannot form 100S ribosomes by deletion of the rmf gene is shorter than that of parental cells under stress conditions in E. coli. This fact indicates that the interconversion system between active 70S ribosomes and inactive 100S ribosomes is an important survival strategy for bacteria. © 2014 John Wiley & Sons, Ltd.


Ueta M.,Yoshida Biological Laboratory | Ueta M.,Osaka Medical College | Wada C.,Yoshida Biological Laboratory | Wada C.,Kyoto University | Wada A.,Yoshida Biological Laboratory
Genes to Cells | Year: 2010

In the stationary growth phase of Escherichia coli, the 70S ribosomes are dimerized by the ribosome modulation factor (RMF) and hibernation promoting factor (HPF) proteins to form 100S ribosomes, which lose translational activity. In this study we found 100S ribosomes in the gram-positive bacterium Staphylococcus aureus, which has an HPF homolog (named SaHPF) but no RMF homolog. Unlike in E. coli, 100S ribosomes exist in all growth phases of S. aureus, with the highest levels at the transition from the exponential phase to the stationary phase. To find the key factors involved in 100S formation, we analyzed proteins associated with crude ribosomes using radical-free and highly reducing 2-D PAGE and MALDI TOF/MS. Only the SaHPF levels changed in parallel with the changes in 100S levels. SaHPF bound preferentially to 70S components in 100S ribosomes, with a molar ratio of 1: 1 relative to the 70S, but some SaHPF was also detected in free 70S ribosomes. High-salt washing of the crude ribosomes released SaHPF and dissociated the 100S ribosomes to their 70S components. When these 70S components were incubated with purified SaHPF in vitro, they re-associated to form 100S. These results suggest that SaHPF is a key protein involved in 100S ribosome formation in S. aureus. © 2009 The Authors. Journal compilation © 2009 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.


PubMed | Yoshida Biological Laboratory
Type: Journal Article | Journal: Genes to cells : devoted to molecular & cellular mechanisms | Year: 2010

In the stationary growth phase of Escherichia coli, the 70S ribosomes are dimerized by the ribosome modulation factor (RMF) and hibernation promoting factor (HPF) proteins to form 100S ribosomes, which lose translational activity. In this study we found 100S ribosomes in the gram-positive bacterium Staphylococcus aureus, which has an HPF homolog (named SaHPF) but no RMF homolog. Unlike in E. coli, 100S ribosomes exist in all growth phases of S. aureus, with the highest levels at the transition from the exponential phase to the stationary phase. To find the key factors involved in 100S formation, we analyzed proteins associated with crude ribosomes using radical-free and highly reducing 2-D PAGE and MALDI TOF/MS. Only the SaHPF levels changed in parallel with the changes in 100S levels. SaHPF bound preferentially to 70S components in 100S ribosomes, with a molar ratio of 1 : 1 relative to the 70S, but some SaHPF was also detected in free 70S ribosomes. High-salt washing of the crude ribosomes released SaHPF and dissociated the 100S ribosomes to their 70S components. When these 70S components were incubated with purified SaHPF in vitro, they re-associated to form 100S. These results suggest that SaHPF is a key protein involved in 100S ribosome formation in S. aureus.

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