Molecular Wiring of a Mitochondrial Translational Feedback Loop.

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.

Spatial orchestration of mitochondrial translation and OXPHOS complex assembly.

Oxidative phosphorylation (OXPHOS) is vital for the regeneration of the vast majority of ATP in eukaryotic cells 1 . OXPHOS is carried out by large multi-subunit protein complexes in the cristae membranes, which are invaginations of the mitochondrial inner membrane. The OXPHOS complexes are a mix of subunits encoded in the nuclear and mitochondrial genomes. Thus, the assembly of these dual-origin complexes is an enormous logistical challenge for the cell. Using super-resolution microscopy (nanoscopy) and quantitative cryo-immunogold electron microscopy, we determined where specific transcripts are translated and where distinct assembly steps of the dual-origin complexes in the yeast Saccharomyces cerevisiae occur. Our data indicate that the mitochondrially encoded proteins of complex III and complex IV are preferentially inserted in different sites of the inner membrane than those of complex V. We further demonstrate that the early, but not the late, assembly steps of complex III and complex IV occur preferentially in the inner boundary membrane. By contrast, all steps of complex V assembly occur mainly in the cristae membranes. Thus, OXPHOS complex assembly is spatially well orchestrated, probably representing an unappreciated regulatory layer in mitochondrial biogenesis.

Organization of Mitochondrial Gene Expression in Two Distinct Ribosome-Containing Assemblies.

Mitochondria contain their own genetic system that provides subunits of the complexes driving oxidative phosphorylation. A quarter of the mitochondrial proteome participates in gene expression, but how all these factors are orchestrated and spatially organized is currently unknown. Here, we established a method to purify and analyze native and intact complexes of mitochondrial ribosomes. Quantitative mass spectrometry revealed extensive interactions of ribosomes with factors involved in all the steps of posttranscriptional gene expression. These interactions result in large expressosome-like assemblies that we termed mitochondrial organization of gene expression (MIOREX) complexes. Superresolution microscopy revealed that most MIOREX complexes are evenly distributed throughout the mitochondrial network, whereas a subset is present as nucleoid-MIOREX complexes that unite the whole spectrum of organellar gene expression. Our work therefore provides a conceptual framework for the spatial organization of mitochondrial protein synthesis that likely developed to facilitate gene expression in the organelle.

Mitochondrial protein synthesis: efficiency and accuracy.

SIGNIFICANCE: The mitochondrial genetic system is responsible for the production of a few core-subunits of the respiratory chain and ATP synthase, the membrane protein complexes driving oxidative phosphorylation (OXPHOS). Efficiency and accuracy of mitochondrial protein synthesis determines how efficiently new OXPHOS complexes can be made. RECENT ADVANCES: The system responsible for expression of the mitochondrial-encoded subunits developed from that of the bacterial ancestor of mitochondria. Importantly, many aspects of genome organization, transcription, and translation have diverged during evolution. Recent research has provided new insights into the architecture, regulation, and organelle-specific features of mitochondrial translation. Mitochondrial ribosomes contain a number of proteins absent from prokaryotic ribosomes, implying that in mitochondria, ribosomes were tailored to fit the requirements of the organelle. In addition, mitochondrial gene expression is regulated post-transcriptionally by a number of mRNA-specific translational activators. At least in yeast, these factors can regulate translation in respect to OXPHOS complex assembly to adjust the level of newly synthesized proteins to amounts that can be successfully assembled into respiratory chain complexes. CRITICAL ISSUES: Mitochondrial gene expression is determining aging in eukaryotes, and a number of recent reports indicate that efficiency of translation directly influences this process. FUTURE DIRECTIONS: Here we will summarize recent advances in our understanding of mitochondrial protein synthesis by comparing the knowledge acquired in the systems most commonly used to study mitochondrial biogenesis. However, many steps have not been understood mechanistically. Innovative biochemical and genetic approaches have to be elaborated to shed light on these important processes.

The Cbp3-Cbp6 complex coordinates cytochrome b synthesis with bc(1) complex assembly in yeast mitochondria.

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.

Cbp3-Cbp6 interacts with the yeast mitochondrial ribosomal tunnel exit and promotes cytochrome b synthesis and assembly.

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.

Ribosome-binding proteins Mdm38 and Mba1 display overlapping functions for regulation of mitochondrial translation.

Biogenesis of respiratory chain complexes depends on the expression of mitochondrial-encoded subunits. Their synthesis occurs on membrane-associated ribosomes and is probably coupled to their membrane insertion. Defects in expression of mitochondrial translation products are among the major causes of mitochondrial disorders. Mdm38 is related to Letm1, a protein affected in Wolf-Hirschhorn syndrome patients. Like Mba1 and Oxa1, Mdm38 is an inner membrane protein that interacts with ribosomes and is involved in respiratory chain biogenesis. We find that simultaneous loss of Mba1 and Mdm38 causes severe synthetic defects in the biogenesis of cytochrome reductase and cytochrome oxidase. These defects are not due to a compromised membrane binding of ribosomes but the consequence of a mis-regulation in the synthesis of Cox1 and cytochrome b. Cox1 expression is restored by replacing Cox1-specific regulatory regions in the mRNA. We conclude, that Mdm38 and Mba1 exhibit overlapping regulatory functions in translation of selected mitochondrial mRNAs.