Abstract
Proving with certainty that a GFP-tagged protein is imported inside mitochondria by visualizing its fluorescence emission with an epifluorescence microscope is currently impossible using regular GFP-tagging. This is particularly true for proteins dual localized in the cytosol and mitochondria, which have been estimated to represent up to one third of the established mitoproteomes. These proteins are usually composed of a surpassingly abundant pool of the cytosolic isoform compared to the mitochondrial isoform. As a consequence, when tagged with a regular GFP, the fluorescence emission of the cytosolic isoform will inevitably eclipse that of the mitochondrial one and prevent the detection of the mitochondrial echoform. To overcome this technical limit, we engineered a yeast strain expressing a new type of GFP called Bi-Genomic Mitochondrial-Split-GFP (BiG Mito-Split-GFP). In this strain, one moiety of the GFP is encoded by the mitochondrial DNA while the second moiety of the GFP can be tagged to any nuclear-encoded protein (suspected to be dual localized or bona fide mitochondrial). By doing so, only mitochondrial proteins or echoforms of dual localized proteins, regardless of their organismal origin, trigger GFP reconstitution that can be visualized by regular fluorescence microscopy. The strength of the BiG Mito-Split-GFP system is that proof of the mitochondrial localization of a given protein rests on a simple and effortless microscopy observation.
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15 November 2022
In Chapter 4, “Assessment of Mitochondrial Complex II and III Activity in Brain Sections: A
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Acknowledgments
This work of the Interdisciplinary Thematic Institute IMCBio, as part of the ITI 2021-2028 program of the University of Strasbourg, CNRS and Inserm, was supported by IdEx Unistra (ANR-10-IDEX-0002), and by SFRI-STRAT’US project (ANR 20- SFRI-0012) and EUR IMCBio (ANR-17-EURE-0023) under the framework of the French Investments for the Future Program (to H.D.B, M.H, B.S); the National Science Center of Poland grant nr UMO-2018-31-B-NZ3-01117 (to R.K); and the Partenariat Hubert Curien Polonium 2021 Program Projet No. 44912NJ.
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Hemmerle, M., Senger, B., di Rago, JP., Kucharczyk, R., Becker, H.D. (2022). Visualizing Mitochondrial Importability of a Protein Using the Yeast Bi-Genomic Mitochondrial-Split-GFP Strain and an Ordinary Fluorescence Microscope. In: Tomar, N. (eds) Mitochondria. Methods in Molecular Biology, vol 2497. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2309-1_16
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DOI: https://doi.org/10.1007/978-1-0716-2309-1_16
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