Abstract
The isolation and functional reconstitution of large membrane protein complexes is an important step towards the biochemical characterization of such sophisticated molecular machines. Reconstitution is a multistep process that requires the mild solubilization of membrane protein complexes from native membrane preparations, the purification of the complexes from protein–detergent solutions, and their incorporation into artificial phospholipid vesicles through controlled detergent removal. The major challenge is to preserve the integrity and catalytic activity of the often fragile membrane protein assemblies during the entire procedure. Here we describe the protocols for a particularly intricate example, the functional reconstitution of the mitochondrial presequence translocase (TIM23 complex). This highly versatile and dynamic protein complex is the main protein translocation machinery of the inner mitochondrial membrane and mediates the import of precursor proteins with N-terminal presequences from the cytosol.
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References
Krogh A, Larsson B, von Heijne G et al (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580
Drews J (2000) Drug discovery: a historical perspective. Science 287:1960–1964
Andreoli TE (1974) Planar lipid bilayer membranes. Methods Enzymol 32:513–539
Reeves JP, Dowben RM (1969) Formation and properties of thin-walled phospholipid vesicles. J Cell Physiol 73:49–60
Darszon A, Vandenberg CA, Schonfeld M et al (1980) Reassembly of protein-lipid complexes into large bilayer vesicles: perspectives for membrane reconstitution. Proc Natl Acad Sci USA 77:239–243
Tribet C, Audebert R, Popot JL (1996) Amphipols: polymers that keep membrane proteins soluble in aqueous solutions. Proc Natl Acad Sci USA 93:15047–15050
Baneres JL, Popot JL, Mouillac B (2011) New advances in production and functional folding of G-protein-coupled receptors. Trends Biotechnol 29:314–322
Heerklotz H, Tsamaloukas AD, Keller S (2009) Monitoring detergent-mediated solubilization and reconstitution of lipid membranes by isothermal titration calorimetry. Nat Protoc 4:686–697
Le Maire M, Champeil P, Moller JV (2000) Interaction of membrane proteins and lipids with solubilizing detergents. Biochim Biophys Acta 1508:86–111
van der Laan M, Meinecke M, Dudek J et al (2007) Motor-free mitochondrial presequence translocase drives membrane integration of preproteins. Nat Cell Biol 9:1152–1159
Neupert W, Herrmann JM (2007) Translocation of proteins into mitochondria. Annu Rev Biochem 76:723–749
Chacinska A, Koehler CM, Milenkovic D et al (2009) Importing mitochondrial proteins: machineries and mechanisms. Cell 138:628–644
Meisinger C, Pfanner N, Truscott KN (2006) Isolation of yeast mitochondria. Methods Mol Biol 313:33–39
Chacinska A, Lind M, Frazier AE et al (2005) Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17. Cell 120:817–829
Knop M, Siegers K, Pereira G et al (1999) Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines. Yeast 15:963–972
Kusters R, Dowhan W, de Kruijff B (1991) Negatively charged phospholipids restore prePhoE translocation across phosphatidylglycerol-depleted Escherichia coli inner membranes. J Biol Chem 266:8659–8662
Ridder AN, Kuhn A, Killian JA et al (2001) Anionic lipids stimulate Sec-independent insertion of a membrane protein lacking charged amino acid side chains. EMBO Rep 2:403–408
Paternostre MT, Roux M, Rigaud JL (1988) Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 1. Solubilization of large unilamellar liposomes (prepared by reverse-phase evaporation) by Triton X-100, octyl glucoside, and sodium cholate. Biochemistry 27:2668–2677
Wittig I, Schägger H (2008) Features and applications of blue-native and clear-native electrophoresis. Proteomics 8:3974–3990
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 746, and the Excellence Initiative of the German Federal and State Governments (EXC 294).
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van der Laan, M., Zerbes, R.M., van der Does, C. (2013). Reconstitution of Mitochondrial Presequence Translocase into Proteoliposomes. In: Rapaport, D., Herrmann, J. (eds) Membrane Biogenesis. Methods in Molecular Biology, vol 1033. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-487-6_21
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DOI: https://doi.org/10.1007/978-1-62703-487-6_21
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