RESUMEN
Mitochondrial respiratory chain complexes convert chemical energy into a membrane potential by connecting electron transport with charge separation. Electron transport relies on redox cofactors that occupy strategic positions in the complexes. How these redox cofactors are assembled into the complexes is not known. Cytochrome b, a central catalytic subunit of complex III, contains two heme bs. Here, we unravel the sequence of events in the mitochondrial inner membrane by which cytochrome b is hemylated. Heme incorporation occurs in a strict sequential process that involves interactions of the newly synthesized cytochrome b with assembly factors and structural complex III subunits. These interactions are functionally connected to cofactor acquisition that triggers the progression of cytochrome b through successive assembly intermediates. Failure to hemylate cytochrome b sequesters the Cbp3-Cbp6 complex in early assembly intermediates, thereby causing a reduction in cytochrome b synthesis via a feedback loop that senses hemylation of cytochrome b.
Asunto(s)
Citocromos b/metabolismo , Hemo/metabolismo , Mitocondrias/genética , Biosíntesis de Proteínas/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citocromos b/genética , Evolución Molecular , Retroalimentación Fisiológica/fisiología , Genes Mitocondriales/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/metabolismo , Oxidación-Reducción , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Respiratory chain complexes in mitochondria are assembled from subunits derived from two genetic systems. For example, the bc(1) complex consists of nine nuclear encoded subunits and the mitochondrially encoded subunit cytochrome b. We recently showed that the Cbp3-Cbp6 complex has a dual function for biogenesis of cytochrome b: it is both required for efficient synthesis of cytochrome b and for protection of the newly synthesized protein from proteolysis. Here, we report that Cbp3-Cbp6 also coordinates cytochrome b synthesis with bc(1) complex assembly. We show that newly synthesized cytochrome b assembled through a series of four assembly intermediates. Blocking assembly at early and intermediate steps resulted in sequestration of Cbp3-Cbp6 in a cytochrome b-containing complex, thereby making Cbp3-Cbp6 unavailable for cytochrome b synthesis and thus reducing overall cytochrome b levels. This feedback loop regulates protein synthesis at the inner mitochondrial membrane by directly monitoring the efficiency of bc(1) complex assembly.
Asunto(s)
Grupo Citocromo b/biosíntesis , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Grupo Citocromo b/metabolismo , Proteínas de la Membrana/genética , Mitocondrias/genética , Proteínas Mitocondriales/genética , Modelos Biológicos , Chaperonas Moleculares/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Mitochondria contain their own genetic system to express a small number of hydrophobic polypeptides, including cytochrome b, an essential subunit of the bc(1) complex of the respiratory chain. In this paper, we show in yeast that Cbp3, a bc(1) complex assembly factor, and Cbp6, a regulator of cytochrome b translation, form a complex that associates with the polypeptide tunnel exit of mitochondrial ribosomes and that exhibits two important functions in the biogenesis of cytochrome b. On the one hand, the interaction of Cbp3 and Cbp6 with mitochondrial ribosomes is necessary for efficient translation of cytochrome b transcript [corrected]. On the other hand, the Cbp3-Cbp6 complex interacts directly with newly synthesized cytochrome b in an assembly intermediate that is not ribosome bound and that contains the assembly factor Cbp4. Our results suggest that synthesis of cytochrome b occurs preferentially on those ribosomes that have the Cbp3-Cbp6 complex bound to their tunnel exit, an arrangement that may ensure tight coordination of cytochrome b synthesis and assembly.
Asunto(s)
Citocromos b/biosíntesis , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de la Membrana/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Chaperonas Moleculares/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transaminasas/genética , Transaminasas/metabolismoRESUMEN
The ribosomal polypeptide tunnel exit is the site where a variety of factors interact with newly synthesized proteins to guide them through the early steps of their biogenesis. In mitochondrial ribosomes, this site has been considerably modified in the course of evolution. In contrast to all other translation systems, mitochondrial ribosomes are responsible for the synthesis of only a few hydrophobic membrane proteins that are essential subunits of the mitochondrial respiratory chain. Membrane insertion of these proteins occurs co-translationally and is connected to a sophisticated assembly process that not only includes the assembly of the different subunits but also the acquisition of redox co-factors. Here, we describe how mitochondrial translation is organized in the context of respiratory chain assembly and speculate how alteration of the ribosomal tunnel exit might allow the establishment of a subset of specialized ribosomes that individually organize the early steps in the biogenesis of distinct mitochondrially-encoded proteins.
Asunto(s)
Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Bacterias/genética , Bacterias/metabolismo , Transporte de Electrón , Evolución Molecular , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Fosforilación Oxidativa , Biosíntesis de Proteínas , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Ribosomas/química , Ribosomas/genéticaRESUMEN
Oxidative phosphorylation in mitochondria requires the synthesis of proteins encoded in the mitochondrial DNA. The mitochondrial translation machinery differs significantly from that of the bacterial ancestor of the organelle. This is especially evident from many mitochondria-specific ribosomal proteins. An important site of the ribosome is the polypeptide tunnel exit. Here, nascent chains are exposed to an aqueous environment for the first time. Many biogenesis factors interact with the tunnel exit of pro- and eukaryotic ribosomes to help the newly synthesized proteins to mature. To date, nothing is known about the organization of the tunnel exit of mitochondrial ribosomes. We therefore undertook a comprehensive approach to determine the composition of the yeast mitochondrial ribosomal tunnel exit. Mitochondria contain homologues of the ribosomal proteins located at this site in bacterial ribosomes. Here, we identified proteins located in their proximity by chemical cross-linking and mass spectrometry. Our analysis revealed a complex network of interacting proteins including proteins and protein domains specific to mitochondrial ribosomes. This network includes Mba1, the membrane-bound ribosome receptor of the inner membrane, as well as Mrpl3, Mrpl13, and Mrpl27, which constitute ribosomal proteins exclusively found in mitochondria. This unique architecture of the tunnel exit is presumably an adaptation of the translation system to the specific requirements of the organelle.
Asunto(s)
Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Péptidos/química , Proteínas Ribosómicas/química , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Membrana Celular/metabolismo , Reactivos de Enlaces Cruzados/química , Microscopía por Crioelectrón/métodos , ADN Mitocondrial/metabolismo , Espectrometría de Masas/métodos , Estrés Oxidativo , Fosforilación , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
Protein tagging with a peptide is a commonly used technique to facilitate protein detection and to carry out protein purification. Flexibility with respect to the peptide tag is essential since no single tag suites all purposes. This report describes the usage of two short peptides from the SARS-associated coronavirus nucleocapsid (SARS-N) protein as protein tags. Plasmids for the generation of tagged proteins were generated by ligating synthetic oligonucleotides for the peptide-coding regions downstream of the protein coding sequence. The data show recognition of prokaryotically expressed HIV-1 Gag/p24 fusion protein by Western blot and efficient affinity purification using monoclonal antibodies against the tags. The SARS peptide antibody system described presents an alternative tagging opportunity in the growing field of protein science.