Structural insights into protein biogenesis on the mitochondrial surface

Abstract

Translation on the surface of the endoplasmic reticulum (ER), as well as co-translational import into the ER have been well characterized, while complementary processes on the mitochondrial surface have remained largely enigmatic. In this thesis we aimed to improve our understanding of the cytosolic ribosome-mitochondria association, polyribosome organization and states of translation on the outer mitochondrial membrane (OMM). The first chapter describes the detailed protocols used for the sample preparation of isolated yeast and human mitochondria from cultured cells and gives an overview over the main method used for this thesis, cryo-ET. In the second chapter we describe the molecular organization of the cytosolic translation machinery on the surface of isolated yeast mitochondria. We present spatial, geometrical, and molecular analysis of ribosomes attached to mitochondria using clustering, subtomogram-averaging and 3D classification approaches. We show that polysome organization adapts to the geometrical constraints of the mitochondrial outer membrane (OMM), but within a flexible regime. Most ribosomes are programmed, whereas only 4 % are found hibernating and connected to the OMM in an unexpected manner. Based on the observation that the conformation of ES27L correlates with the presence of OMM density in subtomogram averages, we visualized a molecular anchor connecting ribosomes to the yeast OMM. A final comparison to published data from Schizosaccharomyces pombe reveals clear species-specific differences and suggests translation on the mitochondrial surface needs to be addressed in several species, and likely differs among different eukaryotes. In the third chapter we build on the toolset used and developed in the second chapter and apply it to cryo-electron tomograms of isolated human mitochondria. In contrast to yeast, the translation arresting drug cycloheximide is not strictly necessary to keep ribosomes attached to mitochondria during isolation. The majority of ribosomes are programmed with tRNAs, unexpectedly with the A-tRNA delivering elongation factor eEF1A still present. The inactive proportion is bound by the elongation factor eEF2 associated with hibernating ribosomes. In contrast to yeast there is no correlation between ES27L conformation and distance or orientation to the OMM, since on the human OMM ribosomes predominantly attach in a tilted manner via ES39L. Comparison to ER-bound ribosomes highlights in total three rRNA expansion segments as organelle specificity facilitating modules in humans. Comparison to less complex eukaryotes furthermore illustrates how ribosomal expansion segments contribute to molecular specialization in higher eukaryotes. In the fourth chapter we describe our efforts to reconstitute the ribosome-mitochondria association observed in the third chapter in vitro with the aim to generate a more defined sample, more suitable for averaging techniques. For initial validation of the mitochondrial sample used in the previous chapters, we established an in vitro import assay using HEK cell mitochondria and radiolabeled in vitro translated mitochondrial precursors. After adaptation of the protocol for cryo-ET, we successfully targeted ribosomes to the outer mitochondrial membrane in vitro. However, we found these ribosomes in an unexpected state and associated in a peculiar manner.