Early stages in the biogenesis of mitochondrial β-barrel proteins

Abstract

Mitochondria are eukaryotic organelles involved in many essential cellular processes. Their functions include the production of energy and the biosynthesis of iron-sulfur clusters and heme. Furthermore, they engage in lipid and amino acid metabolism, signaling pathways, and apoptosis. Considering this functional diversity, it is not surprising that mitochondrial dysfunctions are implicated in neurodegenerative disorders like Parkinson’s and Alzheimer’s disease, in the development of cancer and in aging. To properly fulfill their roles, mitochondria harbor a diverse set of proteins. However, most of these are encoded in the nucleus, translated on cytosolic ribosomes, and are then imported into the organelle. To date, we have a detailed understanding of the intra-organellar stages of the mitochondrial import process. In contrast, little is known about the early, pre-mitochondrial events in the biogenesis of mitochondrial precursor proteins. To better understand the processes in the cytosol that precede the mitochondrial import, I analyzed such early events as part of the biogenesis of β-barrel proteins. These proteins are imported and assembled into the mitochondrial outer membrane by a well-defined pathway. However, so far, it is not clear how they reach this membrane in the first place. In my study, I examined the nature of the targeting signal that directs β-barrel proteins to mitochondria. The results demonstrate that a dedicated β-hairpin motif is necessary and sufficient for the mitochondrial targeting of these proteins. Such a β-hairpin motif is composed of two β-strands connected by a short loop and it can form a β-sheet with one very hydrophobic face. Moreover, in this study, I could identify Tom20 as the major import receptor that recognizes this signal. Next, I analyzed the interactions of newly synthesized β-barrel proteins with cytosolic chaperones. I identified a set of chaperones and co-chaperones that bind to β-barrel proteins in the cytosol. Furthermore, I could demonstrate that these chaperone/β-barrel protein interactions are required for optimal mitochondrial import of the latter. Of note, a β-hairpin element that can serve as a β-barrel protein targeting signal is sufficient for the recognition by the cytosolic (co-)chaperones. Collectively, my findings allow us to outline the early, cytosolic events in the biogenesis of mitochondrial β-barrel proteins. Upon their synthesis on cytosolic ribosomes, these proteins associate with molecular chaperones that keep them in an import-competent conformation. The β-barrel proteins are then targeted to mitochondria via the docking of bound chaperones on the import receptor Tom70 as well as through the interaction of Tom20 with their targeting signal in the form of a dedicated β-hairpin motif.