RESUMO
Proximity-dependent biotin identification (BioID) permits biotinylation of proteins interacting directly, indirectly, or just localized in proximity of a protein of interest (bait). Here, we describe how BioID coupled to proteomics and network biology can be used to map protein proximities in yeast mitochondria, aiding in visualization of complex protein-protein interaction landscapes. For complete information on the use and execution of this protocol, please refer to Singh et al., 2020.
Assuntos
Mitocôndrias/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteômica/métodos , Biotina/química , Biotina/metabolismo , Biotinilação/métodos , Biologia Computacional/métodos , Mitocôndrias/fisiologia , Ligação Proteica/fisiologia , Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Mitochondria contain their own gene expression systems, including membrane-bound ribosomes dedicated to synthesizing a few hydrophobic subunits of the oxidative phosphorylation (OXPHOS) complexes. We used a proximity-dependent biotinylation technique, BioID, coupled with mass spectrometry to delineate in baker's yeast a comprehensive network of factors involved in biogenesis of mitochondrial encoded proteins. This mitochondrial gene expression network (MiGENet) encompasses proteins involved in transcription, RNA processing, translation, or protein biogenesis. Our analyses indicate the spatial organization of these processes, thereby revealing basic mechanistic principles and the proteins populating strategically important sites. For example, newly synthesized proteins are directly handed over to ribosomal tunnel exit-bound factors that mediate membrane insertion, co-factor acquisition, or their mounting into OXPHOS complexes in a special early assembly hub. Collectively, the data reveal the connectivity of mitochondrial gene expression, reflecting a unique tailoring of the mitochondrial gene expression system.
Assuntos
Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/genética , Fosforilação Oxidativa , Biossíntese de Proteínas/genética , Saccharomyces cerevisiae/genéticaRESUMO
The mitochondrial oxidative phosphorylation system comprises complexes assembled from subunits derived from mitochondrial and nuclear gene expression. Both genetic systems are coordinated by feedback loops, which control the synthesis of specific mitochondrial encoded subunits. Here, we studied how this occurs in the case of cytochrome b, a key subunit of mitochondrial complex III. Our data suggest the presence of a molecular rheostat consisting of two translational activators, Cbp3-Cbp6 and Cbs1, which operates at the mitoribosomal tunnel exit to connect translational output with assembly efficiency. When Cbp3-Cbp6 is engaged in assembly of cytochrome b, Cbs1 binds to the tunnel exit to sequester the cytochrome b-encoding mRNA, repressing its translation. After mediating complex III assembly, binding of Cbp3-Cbp6 to the tunnel exit replaces Cbs1 and the bound mRNA to permit cytochrome b synthesis. Collectively, the data indicate the molecular wiring of a feedback loop to regulate synthesis of a mitochondrial encoded protein.
Assuntos
Regulação da Expressão Gênica , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Citocromos b/biossíntese , Citocromos b/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Chaperonas Moleculares/metabolismo , RNA Mensageiro/análise , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/metabolismoRESUMO
The oxidative phosphorylation system contains four respiratory chain complexes that connect the transport of electrons to oxygen with the establishment of an electrochemical gradient over the inner membrane for ATP synthesis. Due to the dual genetic source of the respiratory chain subunits, its assembly requires a tight coordination between nuclear and mitochondrial gene expression machineries. In addition, dedicated assembly factors support the step-by-step addition of catalytic and accessory subunits as well as the acquisition of redox cofactors. Studies in yeast have revealed the basic principles underlying the assembly pathways. In this review, we summarize work on the biogenesis of the bc1 complex or complex III, a central component of the mitochondrial energy conversion system.
