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. 2022:2363:165-181.
doi: 10.1007/978-1-0716-1653-6_13.

Assessment of Mitochondrial Protein Topology and Membrane Insertion

Kerstin Schäfer  1 , Carina Engstler  1 , Korbinian Dischinger  1 , Chris Carrie  2   3
Affiliations

Assessment of Mitochondrial Protein Topology and Membrane Insertion

Kerstin Schäfer et al. Methods Mol Biol. 2022.

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.

Keywords: Carbonate extraction; In vivo GFP localization; Membrane integration; Membrane protein topology; Membrane solubilization; Mitochondrial membrane proteins; Mitoplasts; Osmotic swelling; Proteinase K digestion; Self-assembling GFP.

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