RESUMO
We studied the role of cytochrome c (CYTc), which mediates electron transfer between Complexes III and IV, in cellular events related with mitochondrial respiration, plant development and redox homeostasis. We analyzed single and double homozygous mutants in both CYTc-encoding genes from Arabidopsis: CYTC-1 and CYTC-2. While individual mutants were similar to wild-type, knock-out of both genes produced an arrest of embryo development, showing that CYTc function is essential at early stages of plant development. Mutants in which CYTc levels were extremely reduced respective to wild-type had smaller rosettes with a pronounced decrease in parenchymatic cell size and an overall delay in development. Mitochondria from these mutants had lower respiration rates and a relative increase in alternative respiration. Furthermore, the decrease in CYTc severely affected the activity and the amount of Complex IV, without affecting Complexes I and III. Reactive oxygen species levels were reduced in these mutants, which showed induction of genes encoding antioxidant enzymes. Ascorbic acid levels were not affected, suggesting that a small amount of CYTc is enough to support its normal synthesis. We postulate that, in addition to its role as an electron carrier between Complexes III and IV, CYTc influences Complex IV levels in plants, probably reflecting a role of this protein in Complex IV stability. This double function of CYTc most likely explains why it is essential for plant survival.
Assuntos
Arabidopsis/enzimologia , Citocromos c/deficiência , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Antioxidantes/metabolismo , Arabidopsis/citologia , Arabidopsis/embriologia , Arabidopsis/genética , Ácido Ascórbico/metabolismo , Respiração Celular , Citocromos c/genética , Eletroforese em Gel Bidimensional , Estabilidade Enzimática , Genes de Plantas/genética , Homozigoto , Mutação/genética , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Sementes/metabolismo , Estresse FisiológicoRESUMO
SDS normally is strictly avoided during Blue native (BN) PAGE because it leads to disassembly of protein complexes and unfolding of proteins. Here, we report a modified BN-PAGE procedure, which is based on low-SDS treatment of biological samples prior to native gel electrophoresis. Using mitochondrial OXPHOS complexes from Arabidopsis as a model system, low SDS concentrations are shown to partially dissect protein complexes in a very defined and reproducible way. If combined with 2-D BN/SDS-PAGE, generated subcomplexes and their subunits can be systematically investigated, allowing insights into the internal architecture of protein complexes. Furthermore, a 3-D BN/low-SDS BN/SDS-PAGE system is introduced to facilitate structural analysis of individual protein complexes without their previous purification.
Assuntos
Proteínas de Arabidopsis/análise , Complexo I de Transporte de Elétrons/análise , Eletroforese em Gel Bidimensional/métodos , Proteínas Mitocondriais/análise , Arabidopsis/metabolismo , Proteínas de Arabidopsis/isolamento & purificação , Complexo I de Transporte de Elétrons/isolamento & purificação , Proteínas Mitocondriais/isolamento & purificação , Proteoma/análise , Proteoma/isolamento & purificação , Proteômica/métodos , Reprodutibilidade dos TestesRESUMO
The fine structure of intact, close-to-spherical mitochondria from the alga Polytomella was visualized by dual-axis cryo-electron tomography. The supramolecular organization of dimeric ATP synthase in the cristae membranes was investigated by averaging subvolumes of tomograms and 3D details at approximately 6 nm resolution were revealed. Oligomeric ATP synthase is composed of rows of dimers at 12 nm intervals; the dimers make a slight angle along the row. In addition, the main features of monomeric ATP synthase, such as the conically shaped F(1) headpiece, central stalk and stator were revealed. This demonstrates the capability of dual-axis electron tomography to unravel details of proteins and their interactions in complete organelles.
Assuntos
Complexos de ATP Sintetase/química , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Membrana Celular/ultraestrutura , Clorófitas/química , Clorófitas/ultraestrutura , Dimerização , Processamento de Imagem Assistida por ComputadorRESUMO
High-resolution protein separation procedures are an important prerequisite for proteome analyses. Classically, protein separations are based on 2D IEF/SDS-PAGE. Unfortunately, this technique only poorly recovers hydrophobic proteins, and it is not compatible with analyses of proteins in native state. Blue-native PAGE represents a powerful alternative. It is based on the careful integration of negative charge into proteins and protein complexes by the anionic wool dye Coomassie blue, and it allows protein analyses under native ("blue-native") conditions. Upon combination with SDS-PAGE for a second gel dimension, protein complexes separated by the blue-native dimension are dissected into their subunits, which form vertical rows of spots on the resulting 2D gels. 2D blue-native/SDS-PAGE ideally complements 2D IEF/SDS-PAGE in proteomics.