Tamara Flohr

• Most mitochondrial proteins are synthesized in the cytosol and subsequently have to be imported into mitochondria. An N-terminal mitochondrial targeting signal (MTS) guides these mitochondrial precursor proteins into the matrix. However, 25% of all mitoribosomal proteins (MRPs) do not have a typical MTS but use an internal targeting information instead. In this study, the model protein Mrp17 (bS6m) was used to investigate the import pathway of such a protein without a typical MTS. Despite its internal targeting signal, Mrp17 still is efficiently imported via the typical presequence pathway into the mitochondrial matrix. Like other matrix proteins, the translocation of Mrp17 depends on the TOM and TIM23 complex. Surprisingly, Mrp17 has a different energy dependency for the translocation compared to other matrix proteins. On one side, Mrp17 is highly dependent on the membrane potential which is built at the inner membrane by the respiratory chain. On the other side, the import motor with its core component the ATP-dependent mtHsp70 is not needed for the translocation of Mrp17 across the OM and is only needed to pass the inner membrane. This differs from other matrix proteins, which accumulate in the cytosol if the import motor is defective. Thereby low ATP levels, depletion of the import motor, or inhibition of the ATP synthase traps Mrp17 but also other MRPs in the intermembrane space (IMS). This Mitochondrial Triage of Precursor proteins (MitoTraP) is not simply the result of a general translocation block at the level of the inner membrane. Rather, MitoTraP specifically directs a defined subgroup of matrix proteins into the IMS, most of which are constituents of the mitochondrial ribosome. Due to the lack of rRNA in the IMS trapped MRPs will likely be degraded. Since non-imported Mrp17 interacts with assembly factors of the 90S pre-ribosome in the nucleolus, MitoTrap presumably acts as a safeguard mechanism by separating MRPs from the cytosolic ribosomal assembly machinery.