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1.
Genes Genet Syst ; 93(1): 21-24, 2018 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-29343666

RESUMO

Mitochondrial ribosomal protein L32 (MrpL32) of Saccharomyces cerevisiae is homologous to the bacterial L32 ribosomal protein. MrpL32 carries an N-terminal mitochondrion-targeting sequence (MTS) and is about 60 amino acid residues longer at the C-terminus. Adding to its function as a leader sequence, the MTS of MrpL32 has been reported to regulate ribosome biogenesis through its processing by m-AAA protease. However, the function of the C-terminal extension (CE) remains totally unknown. Therefore, we constructed a series of C-terminally truncated mrpl32 (mrpl32ΔC) genes and expressed them in a Δmrpl32 mutant to examine their function. Interestingly, some MrpL32ΔC derivatives exhibited temperature-sensitive (ts) growth on medium with non-fermentable carbon sources. Furthermore, the CE domain of MrpL32, expressed separately from MrpL32ΔC, could rescue the ts phenotype of mutants by improving mitochondrial protein synthesis.


Assuntos
Mitocôndrias/metabolismo , Mutação , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Regiões 5' não Traduzidas , Meios de Cultura , Metaloendopeptidases/metabolismo , Proteínas Mitocondriais/metabolismo , Biossíntese de Proteínas , Domínios Proteicos , Sinais Direcionadores de Proteínas , Proteínas Ribossômicas/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Temperatura
2.
Microbiol Immunol ; 60(6): 407-17, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27080060

RESUMO

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multifunctional protein that is involved in the HCV life cycle and pathogenesis. In this study, a host protein(s) interacting with NS5A by tandem affinity purification were searched for with the aim of elucidating the role of NS5A. An NS5A-interacting protein, SET and MYND domain-containing 3 (SMYD3), a lysine methyltransferase reportedly involved in the development of cancer, was identified. The interaction between NS5A and SMYD3 was confirmed in ectopically expressing, HCV RNA replicon-harboring and HCV-infected cells. The other HCV proteins did not bind to SMYD3. SMYD3 bound to NS5A of HCV genotypes 1b and 2a. Deletion mutational analysis revealed that domains II and III of NS5A (amino acids [aa] 250 to 447) and the MYND and N-SET domains of SMYD3 (aa 1 to 87) are involved in the full extent of NS5A-SMYD3 interaction. NS5A co-localized with SMYD3 exclusively in the cytoplasm, thereby inhibiting nuclear localization of SMYD3. Moreover, NS5A formed a complex with SMYD3 and heat shock protein 90 (HSP90), which is a positive regulator of SMYD3. The intensity of binding between SMYD3 and HSP90 was enhanced by NS5A. Luciferase reporter assay demonstrated that NS5A significantly induces activator protein 1 (AP-1) activity, this being potentiated by co-expression of SMYD3 with NS5A. Taken together, the present results suggest that NS5A interacts with SMYD3 and induces AP-1 activation, possibly by facilitating binding between HSP90 and SMYD3. This may be a novel mechanism of AP-1 activation in HCV-infected cells.


Assuntos
Hepacivirus/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Fator de Transcrição AP-1/biossíntese , Fator de Transcrição AP-1/metabolismo , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular , Citoplasma/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Hepacivirus/genética , Hepatite C/virologia , Histona-Lisina N-Metiltransferase/biossíntese , Interações Hospedeiro-Patógeno , Humanos , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Replicon/fisiologia , Análise de Sequência de Proteína , Deleção de Sequência , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/genética , Replicação Viral/fisiologia
3.
Microbiol Immunol ; 59(11): 700-4, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26454142

RESUMO

The function of inner membrane protein YciB in Escherichia coli has not been identified. In this study, the membrane topology of the protein that contains five transmembrane domains was clarified. YciB was found to interact with various proteins involved in cell elongation and cell division using a bacterial two-hybrid system. It was also found that the deletion mutant of yciB is susceptible to the low osmolarity. These observations together with previous reports indicate that YciB is involved in synthesis of the cell envelope by interacting with cell elongation and cell division complexes.


Assuntos
Divisão Celular/fisiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Domínios e Motivos de Interação entre Proteínas/genética , Deleção de Sequência/fisiologia , Escherichia coli , Técnicas do Sistema de Duplo-Híbrido
4.
Genes Cells ; 20(11): 956-65, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26391555

RESUMO

Escherichia coli propagates by undergoing cycles of lateral elongation, septum formation, and cell fission at the mid-cell. A large number of genes involved in these processes have been identified, but it is likely that others remain. A deletion mutant of yciB (ΔyciB) is shorter in the cell length compared to wild type and, in contrast, over-expression of yciB causes elongation of the cell. Furthermore, the septum localization of ZipA, an essential protein of cell division, is disturbed in a ΔyciB mutant. Purified YciB protein directly interacted with ZipA, which might indicate that YciB is involved in the cell envelope synthesis directed by ZipA in a PBP3-independent manner.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Geraniltranstransferase/genética , Geraniltranstransferase/metabolismo , Sequência de Aminoácidos , Divisão Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo
6.
ISRN Mol Biol ; 2012: 304021, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-27335665

RESUMO

Depletion of YhcB, an inner membrane protein of Escherichia coli, inhibited the growth of rodZ deletion mutant showing that the loss of both YhcB and RodZ is synthetically lethal. Furthermore, YhcB was demonstrated to interact with RodZ as well as several other proteins involved in cell shape maintenance and an inner membrane protein YciS of unknown function, using bacterial two-hybrid system. These observations seem to indicate that YhcB is involved in the biogenesis of cell envelope and the maintenance of cell shape together with RodZ.

7.
Proc Natl Acad Sci U S A ; 108(8): 3199-203, 2011 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-21300907

RESUMO

Elongation factor 4 (EF4) is one of the most conserved proteins present in bacteria as well as in mitochondria and chloroplasts of eukaryotes. Although EF4 has the unique ability to catalyze the back-translocation reaction on posttranslocation state ribosomes, the physiological role of EF4 remains unclear. Here we demonstrate that EF4 is stored at the membrane of Escherichia coli cells and released into the cytoplasm upon conditions of high ionic strength or low temperature. Under such conditions, wild-type E. coli cells overgrow mutant cells lacking the EF4 gene within 5-10 generations. Elevated intracellular Mg(2+) concentrations or low temperature retard bacterial growth and inhibit protein synthesis, probably because of formation of aberrant elongating ribosomal states. We suggest that EF4 binds to these stuck ribosomes and remobilizes them, consistent with the EF4-dependent enhancement (fivefold) in protein synthesis observed under these unfavorable conditions. The strong selective advantage conferred by the presence of EF4 at high intracellular ionic strength or low temperatures explains the ubiquitous distribution and high conservation of EF4.


Assuntos
Proteínas de Escherichia coli/metabolismo , Magnésio , Biossíntese de Proteínas/genética , Fatores de Elongação da Transcrição/metabolismo , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/fisiologia , Magnésio/farmacologia , Concentração Osmolar , Fatores de Iniciação de Peptídeos , Transporte Proteico , Ribossomos/patologia , Temperatura , Fatores de Elongação da Transcrição/fisiologia
8.
FEMS Microbiol Lett ; 309(1): 35-42, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20528937

RESUMO

RodZ (YfgA) is a membrane protein well conserved among bacterial species and important in the determination of cell shape and motility, although the molecular mechanism involved is not well established. We have characterized a DeltarodZ mutant and show that defective peptidoglycan synthesis might be the primary effect of the deletion. A motile pseudorevertant of DeltarodZ isolated possessed a near rod-shaped cell morphology, indicating that RodZ is not absolutely required for the elongation of the lateral cell wall and the synthesis of functional flagella.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Mutação , Parede Celular/genética , Parede Celular/metabolismo , Proteínas do Citoesqueleto/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Flagelos/genética , Flagelos/metabolismo , Dados de Sequência Molecular , Peptidoglicano/metabolismo
9.
DNA Res ; 14(6): 237-46, 2007 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-18180259

RESUMO

Biofilm forming cells are distinctive from the well-investigated planktonic cells and exhibit a different type of gene expression. Several new Escherichia coli genes related to biofilm formation have recently been identified through genomic approaches such as DNA microarray analysis. However, many others involved in this process might have escaped detection due to poor expression, regulatory mechanism, or genetic backgrounds. Here, we screened a collection of single-gene deletion mutants of E. coli named 'Keio collection' to identify genes required for biofilm formation. Of the 3985 mutants of non-essential genes in the collection thus examined, 110 showed a reduction in biofilm formation nine of which have not been well characterized yet. Systematic and quantitative analysis revealed the involvement of genes of various functions and reinforced the importance in biofilm formation of the genes for cell surface structures and cell membrane. Characterization of the nine mutants of function-unknown genes indicated that some of them, such as yfgA that genetically interacts with a periplasmic chaperone gene surA together with yciB and yciM, might be required for the integrity of outer membrane.


Assuntos
Biofilmes , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Genoma Bacteriano/genética , Genômica/métodos , Proteínas de Escherichia coli/genética
10.
FEMS Microbiol Lett ; 254(1): 157-64, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16451194

RESUMO

The mitochondrial ribosome (mitoribosome) has highly evolved from its putative prokaryotic ancestor and varies considerably from one organism to another. To gain further insights into its structural and evolutionary characteristics, we have purified and identified individual mitochondrial ribosomal proteins of Neurospora crassa by mass spectrometry and compared them with those of the budding yeast Saccharomyces cerevisiae. Most of the mitochondrial ribosomal proteins of the two fungi are well conserved with each other, although the degree of conservation varies to a large extent. One of the N. crassa mitochondrial ribosomal proteins was found to be homologous to yeast Mhr1p that is involved in homologous DNA recombination and genome maintenance in yeast mitochondria.


Assuntos
Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Neurospora crassa/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/isolamento & purificação , Humanos , Espectrometria de Massas , Neurospora crassa/genética , Neurospora crassa/ultraestrutura , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
FEMS Microbiol Lett ; 225(1): 107-14, 2003 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-12900028

RESUMO

To understand the nature and function of bacterial biofilm and the process of its formation, we have performed systematic screening of a complete set of Escherichia coli genes/open reading frames (ORFs) to identify those that affect biofilm development upon over-expression. In contrast to the biofilm of strain AG1 used as a control, some of the genes/ORFs when over-expressed led to the formation of an abnormal biofilm such as thin, mat-like, filamentous or one easily detaching from various surfaces. Disruptants of selected genes were constructed in order to clarify their roles in the different stages of biofilm formation. Our results suggest that diverse metabolic pathways contribute to the development of biofilm.


Assuntos
Biofilmes/crescimento & desenvolvimento , Escherichia coli/genética , Escherichia coli/fisiologia , Genes Bacterianos , Proteínas da Membrana Bacteriana Externa/genética , Fímbrias Bacterianas/genética , Expressão Gênica , Mutagênese , Fases de Leitura Aberta , Fenótipo
12.
Eur J Biochem ; 269(21): 5203-14, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12392552

RESUMO

Mitochondrial ribosomal proteins (mrps) of the budding yeast, Saccharomyces cerevisiae, have been extensively characterized genetically and biochemically. However, the list of the genes encoding individual mrps is still not complete and quite a few of the mrps are only predicted from their similarity to bacterial ribosomal proteins. We have constructed a yeast strain in which one of the small subunit proteins, termed Mrp4, was tagged with S-peptide and used for affinity purification of mitochondrial ribosome. Mass spectrometric analysis of the isolated proteins detected most of the small subunit mrps which were previously identified or predicted and about half of the large subunit mrps. In addition, several proteins of unknown function were identified. To confirm their identity further, we added tags to these proteins and analyzed their localization in subcellular fractions. Thus, we have newly established Ymr158w (MrpS8), Ypl013c (MrpS16), Ymr188c (MrpS17) and Ygr165w (MrpS35) as small subunit mrps and Img1, Img2, Ydr116c (MrpL1), Ynl177c (MrpL22), Ynr022c (MrpL50) and Ypr100w (MrpL51) as large subunit mrps.


Assuntos
Mitocôndrias/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Proteínas Ribossômicas/química , Ribossomos/química , Sequência de Aminoácidos , Sequência de Bases , Substâncias Macromoleculares , Espectrometria de Massas , Proteínas Mitocondriais , Dados de Sequência Molecular , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/isolamento & purificação , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Homologia de Sequência de Aminoácidos
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