The mitoribosome as a hub for integrating mitochondrial gene expression : Regulation of translation and early assembly of cytochrome b

Sammanfattning: The mitochondrial proteome is a mosaic of proteins from two different genetic systems. The majority of mitochondrial proteins are encoded in the nuclear genome and are synthesized in the cytosol before imported to mitochondria. Interestingly, a handful of mitochondrial proteins are still encoded in the small mitochondrial genome, which is a remnant of its bacterial origin. These proteins are almost exclusively highly hydrophobic core subunits of the respiratory chain and ATP synthase. To synthesize these proteins, mitochondria contain its own complete gene expression system including factors for replication, transcription and translation. During the course of evolution, these machineries have diverged from its origin and have acquired organelle-specific characteristics. Mitochondrially encoded proteins are synthesized on the specialized and membrane-tethered mitoribosome. To avoid stoichiometric problems during assembly, translation in mitochondria needs to be coordinated with import of nuclear encoded subunits. However, the mechanisms behind regulation of mitochondrial protein biogenesis are still largely unclear.My research has focused on mitochondria in the yeast Saccharomyces cerevisiae. This provides a possibility to manipulate genes in both the nuclear and mitochondrial genomes to study effects on mitochondrial translation and respiration. In this thesis, I present results that contributes to our understanding of how translation regulation is connected to early assembly of mitochondrially encoded proteins. Using a proximity labelling technique called Bio-ID, we constructed a proximity network of proteins covering the whole mitochondrial gene expression system. This revealed a close association of factors involved in transcription, translation, membrane insertion and assembly, with the mitoribosome as a central hub. In particular, the mitoribosomal polypeptide tunnel exit was found to host factors involved in both regulation of translation and early assembly of nascent polypeptides. Further analysis of the tunnel exit proximity interactome resulted in identification of the membrane protein Mrx4 as a novel repressor of cytochrome b (Cytb) synthesis. Cytb is a catalytic subunit of the respiratory complex III and is encoded by the gene COB on the mtDNA. Translation of COB is under control of the regulatory proteins Cbp1, Cbs1, Cbs2, and Cbp3-Cbp6. Mrx4 is shown to interact with Cbs2, likely to sequester COB mRNA at the tunnel exit in a repressed state, until binding of Cbp3-Cbp6 releases Cbs2 and triggers activation of COB translation. Cbp3-Cbp6 then interacts with the newly synthesized Cytb, as it emerges from the tunnel exit, to stabilize the nascent polypeptide in an early assembly intermediate. The thesis further describes this chaperone function of Cbp3-Cbp6 on Cytb using structural models and site-specific photo-crosslinking. The results indicated that conformational changes in Cytb, upon incorporation of heme bH, releases Cbp3-Cbp6 to activate a new round of COB translation.In summary, the work presented in this thesis demonstrates a high level of organization and novel connectivity of mitochondrial gene expression. In addition, it expands on our knowledge regarding the intricate feedback loop regulation of Cytb biogenesis and the importance to coordinate mitochondrial translation with import of nuclear encoded subunits.