Analysis of the structure of the Oxa1-ribosome-complex by high-resolution cyro-electron microscopy

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Eukaryotic cells contain two translation machineries, one in the cytosol and one in mitochondria. While the cytosolic machinery attracted lots of attention in the past, the mitochondrial machinery is still poorly characterized. Initial studies indicate that mitochondrial ribosomes are surprisingly different from those of the eukaryotic or the bacterial cytosol. This is caused on the one hand by the rapid genetic drift of the rRNA-encoding mitochondrial genome and on the other hand by the specialization of the mitochondrial translation system on the synthesis of a very small number of hydrophobic membrane proteins. Presumably as a consequence of this specialization, the mitochondrial ribosome is intimately associated with the inner membrane and directly binds to Oxa1 and Mba1, two central components of the protein insertion machinery of the inner membrane. The aim of this project is to analyze the structure and function of Oxa1-Mba1- ribosome complexes of fungal mitochondria. During the previous funding period we established procedures to purify ribosomes from Neurospora crassa; cryo-electron microscopy of these particles revealed that this mitochondrial ribosome acquired a significant shell of additional protein. Several of these additional proteins were identified and mapped to the proximity of the polypeptide-exit tunnel. Moreover, we determined the ribosomal binding partners of Oxa1 and Mba1. In the following funding period we will assess the structural and functional properties of the machinery that mediates co-translational protein insertion into the inner membrane, and by this obtain fundamental insights into the specific features of the mitochondrial translation system.