Project Details
Projekt
Mechanisms of mitochondrial proximal translation and co-translational protein import
Applicants
Dr. Yury Bykov; Dr. Simone Mattei
Subject Area
Biochemistry
Term
since 2024
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 541626282
The vast majority of mitochondrial proteins are encoded in the nuclear genome and are synthesized in the cytosol as precursors that must be imported into the mitochondria. While most precursors are completely synthesized in the cytosol and imported post-translationally, for some mitochondrial proteins the translation and translocation events are coupled, resulting in co-translational import. Both import pathways are tightly connected with cytosolic proteostasis and quality control mechanisms. However, it still remains unknown how proteins are sorted between post- and co-translational import pathways and what are the structural elements coupling translation and protein import at the mitochondrial membrane. In this proposal, we aim to address these questions with a synergistic combination of yeast genetics and in situ cryogenic electron tomography (cryo-ET). First, we will use a novel in vivo biotinylation assay to determine which proteins are imported co-translationally. This assay relies on detecting proteins’ biotinylation level, which is proportional to the time spent by the precursors in the cytosol prior to mitochondrial import. Combined with high-throughput yeast genetics, this approach allows rapid analysis of multiple precursors. Second, we will find the determinants of co-translational import and mechanisms of their recognition using structure-function analysis and yeast genetics. To this end, we will visualize ribosomes on mitochondrial membranes in their native cellular context using in situ cryo-ET to study how their numbers, positions, and orientations are regulated in different conditions, complementing this data with in vivo assays. Then, we will use yeast genetics to perturb the primary regulators of ribosome association with the mitochondrial membrane to get a mechanistic understanding of this process. Finally, we will use proteomics approaches to identify proteins that directly interact with ribosomes actively translating precursors targeted to mitochondria. We will then reconstitute these complexes in vitro and will use structural and functional analysis to understand the mechanisms of how mitochondrial protein translation is regulated and coupled to protein import. Our proposal connects the field of translation regulation and quality control with the field of mitochondria biogenesis and thus is integral to the scope of the SPP.
DFG Programme
Priority Programmes
