Final Activity Report Summary - MITOBIO (Mitochondrial biogenesis: evolution of molecular machines) The major aim of this project was to uncover molecular evolution in the pathways mediating the transport of proteins into mitochondria. These organelles are archetypal to eukaryotes and they play essential role in the energy metabolism and programmed cell death for organisms like humans. It seems now that even the most primitive single-celled eukaryotes have mitochondria, although dramatic evolutionary processes transformed the organelles into a very diverse family of relatives. Mitochondria evolved from a class of alphaproteobacteria, which are now common endosymbiotic or parasitic bacteria surviving within eukaryotic cells. As a consequence of the transformation of a bacterium into an organelle, mitochondrial proteins are translated in the cytosol and then transported across two mitochondrial membranes. During the mitochondrial evolution these pathways partially recycled the pre-existing bacterial pathways but mainly created new components, which served in importing all types of mitochondrial proteins. In modern mitochondria, these components assembled into so-called molecular machines which were tuned for a specific subgroup of substrate proteins. Importantly, different mitochondria had different arrangement of these machines but, as we showed, they all shared the minimal complex of subunits. In different evolutionary lineages different sets of accessory subunits completed the molecular machines. On the other hand, single-cell eukaryotes, which inhabited oxygen low environments, reduced their mitochondria to such an extent that they expressed just a handful of proteins. Consequently, these tiny mitochondria, called mitosomes or hydrogenosomes contained only rudiments of the import pathways. Nevertheless, we demonstrated that even these primitive mitochondria, which were present for instance in important human parasites like giardia intestinalis and entamoeba histolytica, still possessed common protein transporting components. As these were extremely divergent in sequence, we had to develop a highly sensitive bioinformatic tool, which first enabled us to discover them among the enormous amount of genomic data which were available. By the combination of in vivo and in vitro experiments we demonstrated that they represented true rudiments of protein transporting machines. These data provided us with the idea about the minimalist functional molecular machines, which might have been present in the first eukaryotic organisms. In addition, these data could offer us the mechanistic details of how these machines worked in a simple set up. Finally, by studying unique biochemical features of the human pathogens we could design more effective chemotherapeutics for the treatment of the infections they caused.