Mitochondria possess their own genome, remnant of the ancestral eubacterial endosymbiont DNA. This mitochondrial genome encodes mostly few key subunits of the respiratory chain. In order to synthesize these few proteins, mitochondria contain a complete gene expression machinery. Crucially, during the evolution, this apparatus dramatically diverged from its bacterial original counterpart, acquiring unique organellar characteristics. Hence, the mechanisms underlying organization and regulation of mitochondrial gene expression are still enigmatic.

In this thesis, I used the model organism Saccharomyces cerevisiae to reveal few aspects of mitochondrial gene expression. Surprisingly, I report that translation initiation strongly diverged from the bacterial one. In fact, the mitochondrial counterpart of the bacterial translation initiation factor 3 is dispensable in yeast. Furthermore, the research made in this work contributed to establish the proximity labelling technique BioID for yeast mitochondrial proteins. This method permitted to analyse extensively the mitochondrial gene expression milieu, creating a comprehensive proximity-based network of factors involved in biogenesis of mitochondrial synthesized proteins. This protein network revealed a unique organization of factors involved in mitochondrial gene expression, meticulously tailored for the synthesis of few organellar proteins. Crucially, we could identify a clear spatial distribution of factors according to their biological function. Moreover, the thesis describes how the polypeptide tunnel exit hosts proteins involved in multiple functions. First, the results show how factors involved in early maturation of Cox1, the core subunit of complex IV of the respiratory chain, reside at the polypeptide tunnel exit. Second, we demonstrate that the synthesis of cytochrome b, subunit of complex III, is also activated at the polypeptide tunnel exit. In fact, proteins taking part in the regulation of mitochondrial gene expression called translational activators interact with this area in an alternate fashion. When synthesis of cytochrome b is repressed, its coding mRNA COB is sequestered at the polypeptide tunnel exit via the binding to Cbs1, a translational activator. The signal that triggers translation initiation is given by Cbp3-Cbp6, a complex that participates in cytochrome b assembly. When a previously synthesized cytochrome b is correctly assembled into complex III, Cbp3-Cbp6 interacts with the polypeptide tunnel exit, forcing the relocation of Cbs1, and making COB mRNA available for a new round of translation. This mechanism represents a unique form of tuning between mitochondrial and nuclear gene expression systems, essential for the correct assembly of complexes made up by proteins of dual origin.

In summary, the work presented in this thesis reveals novel features of the organization and regulation of the mitochondrial gene expression, highlighting many distinctive organellar features. The concepts and techniques presented here will be a starting point to elucidate many unknown aspects of mitochondrial protein synthesis.