Organizing the cell
Organelles are universal building blocks of eukaryotic cells. These are compartments with defined functions and protein compositions. We are interested in the mechanisms that help cells to maintain this order and ship every protein to the correct place. For this, each protein has a special address tag called targeting sequence embedded directly into their amino acid order. Targeting sequences are recognized by targeting factors and translocons that help proteins to get into their destination organelle. We want to understand how the targeting pathways effectively function in living cells where they need to simultaneously sort thousands of proteins to multiple destinations. We are also interested in how the fundamental cellular processes of protein synthesis and folding are connected to protein transport and translocation across membranes. Our favorite organelle is mitochondria, the central hub of cellular metabolism and signaling.
Do mitochondria have co-translational protein import?
The endoplasmic reticulum is famously known to import a lot of proteins co-translationally. Ribosomes tightly bind Sec61 translocon and directly hand over nascent proteins to the ER. Whether this happens on the mitochondrial surface remains unknown. Hundreds of proteins need to be imported and most of them are believed to be completely made in the cytosol and imported into the organelle before folding. However, it is clear some mRNAs and ribosomes do localize to the surface of mitochondria. The mechanisms of this localization as well as the role of this process in mitochondrial biogenesis is unknown. We want to solve this question by observing protein import and ribosome positions in intact cells. For this we employ yeast genetics and collaborate with the group of Dr. Simone Mattei (EMBL Heidelberg).
Global analysis of local translation
While it is well accepted that some mitochondrial proteins are made next to the ER and mitochondria, very little is known about local regulation of translation in other cellular regions. Only recently it became clear that cytosolic ribosomes and mRNAs are not positioned randomly, but translation can be organized by cellular ultrastructure. We plan to construct a comprehensive map of local translation by combining yeast genetics, high-throughput fluorescent microscopy, and in situ cryogenic tomography. We want to know how protein synthesis is connected to protein targeting and how targeting can fine tune translation to enable organelle biogenesis.
Complex life of precursor proteins
Mitochondria import most of their proteins post-translationally. Before import, these proteins spend some time in the cytosol in unfolded form that is called a precursor. There they are bound to chaperones, the guardians of cellular proteostasis. (Proteostasis is key cellular process that is disturbed in disease and ageing!) Relatively quickly, chaperones hand over the precursors to the mitochondrial import machinery. Do some proteins spend more time in the cytosol than the others? What exact chaperones do they bind? What are the specificity determinants? We want to answer these questions by studying transient protein-protein interactions that precursors experience on their way to mitochondria. To study this delicate process, we detect protein-protein interactions right in the living cells.
Protein targeting efficiency
When we ship a parcel by mail, the address on it is precise and unambiguous. Compared to this, protein targeting sequences are rather ambiguous. However, the cells interpret them very effectively and place hundreds of different proteins in exact locations they belong. Do they make mistakes? How do they correct them? How protein localization to several different places is maintained? We want to answer these questions by using an elegant genetic system developed by the groups of Prof. Hubert Becker and Prof. Róża Kucharczyk. They managed to encode part of split GFP directly in the mitochondrial DNA. This system allows to detect fluorescence of only the protein fraction that reaches mitochondrial matrix. Using this approach, we can figure out how precise protein targeting is and how are proteins sorted to different destinations within mitochondria themselves.