Saskia Rödl

• Mitochondrial precursor proteins need to master a complex journey from the cytosol to their final destination within mitochondria. Several hundreds of proteins are simultaneously targeted to and imported into mitochondria by a vast variety of import pathways. To ensure their proper biogenesis, mitochondria closely collaborate with numerous cytosolic factors involving components of the chaperone network and the ubiquitin-proteasome system (UPS). This study aimed to uncover novel mechanisms by which cells monitor proper mitochondrial biogenesis. On the one hand, I elucidated in how far the great versatility of N-terminal presequences contribute to the orderly process of protein import. By fusing several presequences of variable lengths to the model protein DHFR, I generated a test set of precursors which only differ in their targeting sequence. In vitro imports and IQ-Compete, a novel assay specifically established for this study, revealed considerable differences in the import efficiencies of these precursors in vitro and in vivo. Investigating the presequences of Oxa1 and Atp5 as representatives of the test set, I found that either denaturation and preincubation, respectively, or deletion of the receptors Tom70/71 only diminished the import efficiency of the Oxa1 presequence. Furthermore, using immunoprecipitation experiments, I identified the cytosolic co-chaperone and tetratricopeptide repeat (TPR) protein TOMM34 to bind to the presequence of Oxa1 but not to that of Atp5 in reticulocyte lysate from rabbits. Since the mammalian protein TOMM34 is not present in yeast, I investigated whether a yeast TPR co-chaperone might be similarly involved in mitochondrial biogenesis instead. I was able to show that the depletion of the co-chaperone Cns1 resulted in reduced protein levels of the two mitochondrial proteins Oxa1 and Mam33, hinting towards previously unknown role of Cns1 in mitochondrial biogenesis. On the other hand, I revealed novel insights into the surveillance of mitochondrial protein import by specific UPS components. A genetic screen in yeast suggests the ubiquitin conjugase Ubc8 to play a role in efficient precursor biogenesis. I could verify this finding by detecting elevated precursor levels in a Ubc8 deletion mutant. Using dynamic SILAC mass spectrometry, I demonstrated that the loss of Ubc8 broadly affects mitochondrial protein levels. In vitro imports allowed me to attribute these observations to impaired import efficiencies into mitochondria isolated from ubc8 deletion cells. By employing proteomic analyses, I found the receptor Tom22 to be the most diminished component among all proteins of the import machineries in ubc8 deletion cells. In line with this, blue native PAGE experiments revealed strongly diminished levels of the receptor Tom22 being assembled into the TOM complex upon the deletion of Ubc8. Taken together, my findings reveal new aspects of the complex interplay of mitochondria with the cytosolic chaperone network and the UPS to enable proper mitochondrial biogenesis. Some presequences specifically recruit the cytosolic co-chaperone TOMM34 which enables highly efficient import via Tom70. Additionally, the ubiquitin conjugase Ubc8 promotes effective assembly of the receptor Tom22 into a fully functional TOM complex and thereby maintains efficient protein import.