RESUMO
Protein synthesis in mitochondria is generally organized in a bacterial-like manner but, at the same time, possesses several unique traits. Translation initiation in mitochondria is regulated by two protein factors, mtIF2 and mtIF3. Previously we demonstrated that Saccharomyces cerevisiae Aim23 protein is an ortholog of IF3 in budding yeast. However, the data on the interactions between Aim23p and other proteins are limited. Here, we demonstrated that Aim23p interacts with the yeast mitochondrial ribosomal small subunit both in vivo and in vitro using co-immunoprecipitation and density gradient sedimentation.
Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Subunidades Ribossômicas Menores/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Centrifugação com Gradiente de Concentração , Imunoprecipitação , Proteínas Mitocondriais , Ribossomos Mitocondriais , Fator de Iniciação 2 em Procariotos , Fator de Iniciação 3 em Procariotos , Proteínas Ribossômicas/metabolismoRESUMO
HU is a most abundant DNA-binding protein in bacteria. This protein is conserved either in its heterodimeric form or in one of its homodimeric forms in all bacteria, in plant chloroplasts, and in some viruses. HU protein non-specifically binds and bends DNA as a hetero- or homodimer and can participate in DNA supercoiling and DNA condensation. It also takes part in some DNA functions such as replication, recombination, and repair. HU does not recognize any specific sequences but shows some specificity to cruciform DNA and to repair intermediates, e.g., nick, gap, bulge, 3'-overhang, etc. To understand the features of HU binding to DNA and repair intermediates, a fast and easy HU proteins purification procedure is required. Here we report overproduction and purification of the HU homodimers. The method of HU purification allows obtaining a pure recombinant non-tagged protein cloned in Escherichia coli. We applied this method for purification of Acholeplasma laidlawii HU and demonstrated that this protein possesses a DNA-binding activity and is free of contaminating nuclease activity. Besides that we have shown that expression of A. laidlawii ihf_hu gene in a slow-growing hupAB E. coli strain restores the wild-type growth indicating that aclHU can perform the basic functions of E. coli HU in vivo.
Assuntos
Acholeplasma laidlawii/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Recombinantes/metabolismo , Acholeplasma laidlawii/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , DNA/genética , DNA/metabolismo , DNA Circular/genética , DNA Circular/metabolismo , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Teste de Complementação Genética , Mutação , Plasmídeos/genética , Plasmídeos/metabolismo , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ressonância de Plasmônio de SuperfícieRESUMO
Plasmid vectors encoding hydrophilic (IncB, IncC, IncE, IncG) and hydrophobic (IncC, IncG) domains of C. trachomatis incorporation membrane proteins and reporter green fluorescing proteins were constructed. After transfection of HeLa cells with these plasmid constructs, localization of the complex proteins was determined by laser confocal microscopy. Tropism of hydrophobic domains to compartments constituting the exocytotic pathway in the cell was demonstrated. Location of signal/sorting sequences responsible for specific localization was determined.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/genética , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Bactérias/química , Células Cultivadas , Regulação Bacteriana da Expressão Gênica , Células HeLa , Humanos , Corpos de Inclusão/metabolismo , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Distribuição Tecidual , TransfecçãoRESUMO
The protein and peptide composition of medicinal leech salivary gland secretion (SGS) was analyzed in preparations obtained in July from three species--Hirudo verbana, H. medicinalis, and H. orientalis. Two-dimensional electrophoresis (molecular mass 10-150 kD and pI 3-10) revealed no distinctions in the distribution of over 100 silver-stained proteins. Differences were noted only in intensity of 10 protein spots at 30-90 kD and pI 4.7-7.5. Mass spectrometric profiling of SGS of the three leech species using the Zip-Tip/golden chip scheme and cation-exchanging chips CM-10 revealed over 50 components in SGS of each of the three leech species. It was noted that 30-40% of the individual masses of the SGS of each leech species fall within the masses present in SGS of at least one of the two other species. This rather small part of the total mass may be indicative of a high polymorphism of amino acid sequences or a high frequency of posttranslational modifications of the SGS proteins and peptides. Calculation of Jacquard's coefficient showed that H. medicinalis and H. orientalis are closest to each other in SGS composition, which is consistent with data in the literature on the phylogenetic relationship between these two species of medicinal leech. Comparison of detected molecular masses with those of six known biologically active compounds produced by medicinal leeches revealed their uneven distribution in SGS of each of the three medicinal leech species. This opens prospects for using certain species of medicinal leech for targeted therapy of various pathologies.
