Die Ausbildung von Kontaktstellen zwischen Peroxisomen und Mitochondrien durch Proteine mit dualen Lokalisierungssignalen

The transport of proteins into membrane-bound organelles, such as mitochondria or peroxisomes, is mediated by specific targeting signals. How these proteins are distributed between two organelles and how this impacts the subcellular organization of organelles is largely elusive. A previous study demonstrated that the phosphatase Ptc5 from Saccharomyces cerevisiae, which possesses an N-terminal signal for mitochondria and a C-terminal signal for peroxisomes, initially inserts into mitochondria, is processed at the inner mitochondrial membrane, and subsequently translocates into peroxisomes. This project focused on the investigation if proteins with targeting signals for peroxisomes and mitochondria act as tethers between these organelles, due to their affinity for both import machineries. Additionally, this project aimed to characterize proteins relevant for the translocation of Ptc5 to peroxisomes. The results probed the hypothesis that direct contact between the two organelles is necessary for this process to occur. It was demonstrated that proteins with dual localization signals induce the formation of organelle contacts. Their overexpression increased the association of peroxisomes with mitochondria, as demonstrated by fluorescence and electron microscopy. I found that organelle association is regulated depending on the metabolic state of the cell. It was induced under conditions where peroxisomes are metabolically active (e. g. in conditions requiring lysine biosynthesis or in the presence of oleic acid as only carbon source). Particularly, proteins carrying two targeting signals, which were efficiently retained in mitochondria acted as strong tethers. The phosphatase Ptc5 from Saccharomyces cerevisiae transits via mitochondria to peroxisomes but has no strong tethering function. A genetic screen identified mutants that impaired peroxisomal localization of Ptc5. Of particular interest was the Δmdm10 mutant. Mdm10 is part of the ERMES complex, which connects the endoplasmic reticulum and mitochondria and facilitates lipid transfer between these organelles. Consistent with previous studies, the results showed a role of ERMES in the formation of contacts between mitochondria and peroxisomes. An artificial tether for these organelles suppressed the peroxisome-specific phenotypes resulting from MDM10 deletion. Peroxisomal localization of Ptc5 was restored in these cells, suggesting that that direct protein transfer from mitochondria to peroxisomes depends on physical contact. ERMES components accumulated at three-way junctions between the ER, peroxisomes, and mitochondria. Whether the ERMES complex itself functions as a molecular tether for peroxisomes, or whether this function is mediated indirectly by ERMES-dependent proteins, or both, has not yet been conclusively clarified. This work showed that in ERMES mutants, many proteins with dual targeting signals mislocalize, probably diminishing their tethering function. This points to an indirect contribution of ERMES for peroxisome–mitochondria tethering. In summary, this work contributed to a better understanding of eukaryotic cell biology. The transit of proteins from mitochondria to peroxisomes was unknown until 2020. This work identified new factors involved in this process and demonstrated that proteins with two targeting signals can function as molecular tethers, revealing a previously unexplored mechanism for the formation of contact sites between organelles.