RESUMO
A bacterially expressed recombinant HClpP protein, the human homologue of Escherichia coli ClpP protease, was used to obtain specific polyclonal antibodies. Those antibodies identify a 26 kDa polypeptide in mitochondrial subcellular fractions of rat and human liver. Immunofluorescence and electron microscopic studies demonstrate that the mammalian homologue of ClpP is located in the mitochondrial matrix with a tendency to be found in association with the inner mitochondrial membrane. An HClpP recombinant protein with a truncated NH2terminus (missing the first 58 amino acid residues) shows a molecular mass of 26 kDa under denaturing conditions. This N-truncated HClpP recombinant protein shows a native molecular mass of 340 kDa that is identical with the native molecular mass of the partially purified protein from rat liver mitochondria. Electron microscopy shows that the N-truncated recombinant HClpP has a ring shape with seven identical morphological units in the periphery, exhibiting a 7-fold symmetry. The native molecular mass and the electron microscopic studies suggest that mitochondrial ClpP is composed of two heptameric rings with 7-fold symmetry, similar to E. coli ClpP.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Mitocôndrias Hepáticas/química , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Adenosina Trifosfatases/análise , Sequência de Aminoácidos , Animais , Anticorpos , Linhagem Celular , Cromatografia em Gel , Endopeptidase Clp , Escherichia coli/química , Escherichia coli/genética , Imunofluorescência , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Microscopia Imunoeletrônica , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/ultraestrutura , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Conformação Proteica , Ratos , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Serina Endopeptidases/análiseRESUMO
Assembly of mammalian 20 S proteasomes from individual subunits is beginning to be investigated. Proteasomes are made of four heptameric rings in the configuration alpha7beta7beta7alpha7. By using anti-proteasome and anti-subunit-specific antibodies, we characterized the processing and assembly of the beta subunit C5. The C5 precursor (25 kDa) remains as a free non-assembled polypeptide in the cell. The conversion of the C5 precursor to mature C5 (23 kDa) occurs concomitantly with its incorporation into 15 S proteasome intermediate and 20 S mature proteasome complexes. This processing is dependent on proteasome activity and takes place in the cytosol. These results are not fully compatible with the hypothesis that postulates that assembly of proteasomes takes place via a "half-proteasome" intermediate that contains one full alpha-ring and one full beta-ring of unprocessed beta subunit precursors.