Abstract
Analyzing the membrane integrity and topology of a mitochondrial protein is essential for truly understanding its function. In this chapter, we demonstrate how to analyze mitochondrial membrane proteins using both an immunological-based assay and an in vivo self-assembling GFP approach. First, immunological approaches to investigate the solubility or membrane association of a protein within mitochondria are described. With this method, we demonstrate how the topology of soluble domains of a membrane-integrated protein can be determined. Using protein-specific antibodies, the localization of the soluble domains of a protein are analyzed by a proteolytic-cleavage approach using proteinase K in mitochondria, outer membrane-ruptured mitochondria, and solubilized mitochondrial membranes. In a second approach, we determine the topology of plant mitochondrial proteins using an in vivo self-assembling GFP localization approach.
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Acknowledgments
This work (CA 1775/1-1) was supported by the Deutsche Forschungsgemeinschaft (DFG) granted to C.C.
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Schäfer, K., Engstler, C., Dischinger, K., Carrie, C. (2022). Assessment of Mitochondrial Protein Topology and Membrane Insertion . In: Van Aken, O., Rasmusson, A.G. (eds) Plant Mitochondria. Methods in Molecular Biology, vol 2363. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1653-6_13
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DOI: https://doi.org/10.1007/978-1-0716-1653-6_13
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