Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Ramadani-Muja, J.
Development and application of fluorescent protein-based probes to determine protein targeting to mitochondria.
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Graz Medical University; 2020. pp. 118
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Graier Wolfgang
- Hallström Seth
- Malli Roland
- Altmetrics:
- Abstract:
- Almost all mitochondrial proteins are encoded by nuclear genes, meaning that all of these proteins have to be imported into mitochondria. Therefore all mitochondrial functions rely on an efficient protein import into the organelle. However, the capability of mitochondria to import proteins can be exceeded, leading to an accumulation, hence mistargeting of nuclear encoded mitochondrial proteins in the cytosol and/or nucleus. Thus, the detection of the targeting efficiency of mitochondrial proteins can act as a sensor of mitochondrial fitness or mitochondrial stress, respectively. Here, we introduce the engineering and application of novel fluorescent protein (FP) based probes, protocols and image analysis for the visualization of the MPI and its defects in single cells under various conditions. Applying the first class of MPI-sensors, where we fused FPs to different mitochondrial targeting sequences (MTS), revealed that cancer cells constantly and very efficiently import very high amounts of proteins with optimized MTS to mitochondria. Additionally our data emphasize a cotranslational import of proteins into mitochondria that might explain the high capacity of mitochondria to take up nuclear-encoded proteins. This approach proved also suitable for the detection of defects in MPI, due to depolarization of mitochondria resulting in an instant accumulation of mitochondria-targeted proteins within the cytosol and nucleus. In an additional attempt we developed novel bi- and tripartite sensors, applying self-complementation split-FP (scSplit-FP) and dimerization-dependent FP (ddFP) technologies. Generation of mitochondrial Sirtuin4 Tripartite Abundance Reporter (mito-STAR), based on scSplit-FP approach allows the quantitative investigation of sirtuin 4 (sirt4), a mitochondrial matrix protein considered to be involved in cellular stress responses. Our data indicate that sirt4 may contribute to the mitochondrial unfolded protein response by its accumulation within the cytosol and nucleus upon cell stress. By applying the ddFP-technology, we developed an additional class of FP-based probes to study the efficiency and activity of MPI. We named these probes mitochondrial Protein Import Analyzers (mito-PRIMA). The mito-PRIMAs are dually targeted bipartite probes that allow the detection of impairments in the MPI machinery by generating fluorescence within the nucleus, when the MPI is defect. Exploiting mito-PRIMA revealed that stimulation of transcription can trigger an overload of the MPI machinery in cancer cells. Altogether, the findings presented in this work emphasize that the novel fluorescent MPI-sensors are suitable to gain mechanistic insights into MPI, its defects and mitochondrial-to-nucleus signaling as well as mitochondrial stress responses under various cellular conditions.