Functional analysis of mitochondrial ribosomes: Biogenesis and Function

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Mitochondria contain a miniature genome that codes for a handful of hydrophobic proteins which are synthesized by membrane-associated mitochondrial ribosomes (mitoribosomes). Defects in the biogenesis or function of the mitochondrial translation machinery compromise respiration and are cause of severe diseases in humans.Mitoribosomes are surprisingly different to ribosomes of the bacterial or eukaryotic cytosol. Mitoribosomes contain 70-80 different protein subunits (73 in baker’s yeast) and thus are similarly complex than their cytosolic counterparts. Genetic screens in yeast identified many ribosome assembly factors, helicases, modifying enzymes and regulatory components in the mitochondria. Nonetheless, how mitoribosomes are assembled is still largely unknown and the topic of this proposal. Here, we apply for the second funding period of the project in which two aims will be addressed.Aim 1 will focus on the import of mitoribosomal proteins. In contrast to other matrix proteins, many mitoribosomal proteins lack N-terminal matrix targeting signals. We recently identified the targeting information in the model protein Mrp17 which is surprisingly different to the features found in presequences. Our preliminary data indicate that Mrp17 embarks on the TOM-TIM23 import pathway that is also used by other matrix proteins, however, it does not make use of the Hsp70-dependent import motor. Hence, Mrp17 import is ATP-independent but requires an extremely high membrane potential of the inner membrane. In this project we will elucidate the molecular mechanisms of the unconventional import process of Mrp17 and other mitoribosomal proteins. On the one hand, we want to characterize the mechanistic details of this ATP-independent protein import pathway. On the other hand, we plan to unravel the physiological consequences of this unconventional import process. To this end, we will develop novel competitive import assays which allow it to compare the import efficiency of different types of mitochondria directly in vitro as well as in vivo. Aim 2 focuses on the processes by which mitochondrial ribosomes are assembled. We developed a system in which mitochondrial ribosomes can be temporarily depleted from mitochondria by down-regulation of the mitochondrial RNA polymerase. Upon re-initiation of transcription, mitochondrial ribosomes are re-built. We will use this synchronized system to monitor the assembly of the large and small subunit of the mitoribosome and stage the assembly process into individual reactions using complexome analysis. Preliminary studies already revealed the accumulation of partially stable assembly intermediates which we will now further characterize by proteomics. Furthermore, we will identify the relevance of assembly factors and modifying enzymes during ribosome biogenesis. Thereby, we expect fundamental insights into the biological processes underlying the biosynthesis and function of mitochondrial protein translation machinery.