Protein sorting to peroxisomes via mitochondria: Characterization of the molecular mechanism and its impact on formation of organelle contact sites.

• Applicant: Dr. Johannes Freitag
• Subject Area: Cell Biology ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 455559921

The protein phosphatase Ptc5 from Saccharomyces cerevisiae contains an N-terminal targeting signal for mitochondria and a C-terminal targeting signal for peroxisomes (PTS1). We recently reported that Ptc5 is sorted to peroxisomes via mitochondria by a novel targeting mechanism (Stehlik et al, 2020). We now will characterize the mechanism in greater detail(1.). In addition, we will focus on the following hypothesis: translocation of proteins into peroxisomes from mitochondria triggers the formation of organelle contact sites via molecular tug-of-war, which results from simultaneous interaction of the cargo protein with the import machineries of both compartments. Thus, protein translocation per se may organelles (2.).1. Elucidation of the molecular mechanism of protein sorting to peroxisomes via mitochondria1.1 Intra-molecular signals required for the novel translocation mechanism will be characterized.1.2 Genes identified in the high-content screen (e.g. ERMES components) will be further investigated to address the roles of their gene products for Ptc5 translocation. In addition, we will examine components of the mitochondrial import machinery, which are often encoded by essential genes.1.3 Finally, we will set up an in vitro reconstitution system that recapitulates our in vivo data.2. Functional consequences of indirect sorting to peroxisomesIn the second part of the project we will elucidate how sorting via competing targeting signals influences the physical interaction of mitochondria and peroxisomes. We have identified several S. cerevisiae proteins showing this domain structure (Stehlik et al, 2020).2.1 The effect of tug-of-war like sorting on the association of peroxisomes and mitochondria will be addressed by studying contact site formation upon depletion of components of the peroxisomal import machinery.2.2 We will analyze the biological function of proteins with competing targeting signals (Cat2, Tes1 and Pxp2) via mutants that are transported to peroxisomes directly without detour. Their effect on contact site formation and metabolite exchange between peroxisomes and mitochondria will be determined.2.3 We will create a switchable tether, which can be used to induce artificial contacts. Strains containing this tether will be analyzed for peroxisome function and protein translocation from mitochondria to peroxisomes.The planned project will help to understand how peroxisomal import via a mitochondrial detour contributes to the interplay of both organelles. We are convinced that the study is important as peroxisomes and mitochondria share many metabolic pathways, but how they physically interact is less understood. Since mammalian proteins are known that contain competing peroxisomal and mitochondrial targeting signals, our project may result in a better understanding of the role of peroxisome and mitochondria interaction in human physiology.

• DFG Programme: Research Grants