Mitochondrial Precursor Proteins in the Cytosol: Identification and Characterization of Signals and Factors that Coordinate Early Steps in Mitochondrial Protein Biogenesis

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Mitochondria are essential organelles of eukaryotic cells. They consist of 800 to 1500 proteins, most of which are synthesized in the cytosol as precursor proteins. Little is known about the reactions in the cytosol by which precursor proteins are maintained in an unfolded, import-competent form, targeted to the mitochondria, recognized by surface receptors and sorted into the specific mitochondrial subcompartments. These early processes of mitochondrial protein targeting are under the surveillance of the proteasome system in order to avoid the accumulation of non-imported precursor proteins in the cytosol, but details are largely elusive.In this project, we want to address two related questions: First, we want to understand which features of the mature regions of presequence-containing precursor proteins support their targeting through the cytosol and which features play a role to direct precursor proteins to their specific locations within mitochondria. Recently, we identified internal matrix targeting signal-like sequences (iMTS-Ls) which bind to the mitochondrial surface receptor Tom70. In the first part of this project we will study the relevance of these iMTS-Ls in more detail, analysing their relevance for precursor targeting to the mitochondrial surface, as well as their role in the topogenesis of inner membrane proteins. Most inner membrane proteins contain characteristic patterns consisting of iMTS-Ls in front of, or following, their transmembrane domains, but the specific functions of these patterns are not clear.The second goal of this project is to understand the cellular response to the cytosolic accumulation of mitochondrial precursor proteins. Preliminary experiments show that increased levels of precursors in the cytosol leads to a strong cellular stress response causing the upregulation of chaperones and of components of the proteolytic system of the cytosol. In parallel, cells reduce the expression of mitochondrial precursor proteins, presumably in order to attenuate their toxic effects in the cytosol. We designed strategies to identify and characterize the signaling cascades that allow the cell to elicit these reactions and have already identified critical transcription factors. Moreover, we intend to identify the components that regulate the degradation of precursor proteins by the proteasome, in order to clear the cytosol from aggregation-prone unfolded preproteins and to study their function in this process. From our studies we expect detailed insights into the reactions that regulate the early steps in mitochondrial protein biogenesis, either by stimulating the efficient import of precursor proteins into mitochondria (part 1) or by removing accumulating precursor proteins by the cytosolic quality control system (part 2).