Abstract
The mitochondrial ADP/ATP carrier (Ancp) has long been a paradigm for studies of the mitochondrial carrier family due to, among other properties, its natural abundance and the existence of specific inhibitors, namely, carboxyatractyloside (CATR) and bongkrekic acid (BA), which lock the carrier under distinct and stable conformations. Bovine Anc1p isolated in complex with CATR in the presence of an aminoxyde detergent (LAPAO) was crystallized and its 3D structure determined. It is the first mitochondrial carrier structure resolved at high resolution (2.2 Å, as reported by Pebay-Peyroula et al. (Nature 426:39–44, 2003)). Analyses revealed a monomer while most of the biochemical studies led to hypothesize Ancp functions as a dimer. To address the structural organization issue, we engineered a mutant of the yeast Ancp that corresponds to a covalent homodimer in view of 3D structure determination. We compare in this chapter the purification yield and quality of the chimera tagged either with six histidines at its C-ter end or nine histidines at its N-ter. We show that, as expected, length and position of the tag are important criteria for qualitative purification. We also discuss the advantages and drawbacks of purifying Ancp either from a natural source or from engineered yeast cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Nury H, Dahout-Gonzalez C, Trézéguet V, Lauquin GJ-M, Brandolin G, Pebay-Peyroula E (2006) Relations between structure and function of the mitochondrial ADP/ATP carrier. Annu Rev Biochem 75:713–741
Trézéguet V, Le Saux A, David C, Gourdet C, Fiore C, Dianoux A, Brandolin G, Lauquin GJ-M (2000) A covalent tandem dimer of the mitochondrial ADP/ATP carrier is functional in vivo. Biochim Biophys Acta 1757:81–93
Fiore C, Trézéguet V, Roux P, Le Saux A, Noël F, Schwimmer C, Arlot D, Dianoux A-C, Lauquin GJ-M, Brandolin G (2000) Purification of histidine-tagged mitochondrial ADP/ATP carrier: influence of the conformational states of the C-terminal region. Protein Expr Purif 19:57–65
Brandolin G, Doussiere J, Gulik A, Gulik-Krywicki T, Lauquin GJM, Vignais PV (1980) Kinetic, binding and ultrastructural properties of the beef heart adenine nucleotide carrier protein after incorporation into phospholipid vesicles. Biochim Biophys Acta 592:592–614
De Marcos Lousa C, Trézéguet V, Dianoux A-C, Brandolin G, Lauquin GJ-M (2002) The human mitochondrial ADP/ATP carriers: kinetic properties and biogenesis of wild type and mutant proteins in the yeast S. cerevisiae. Biochemistry 41:14412–14420
Postis V, De Marcos Lousa C, Arnou B, Lauquin GJ-M, Trézéguet V (2005) Subunits of the yeast mitochondrial ADP/ATP carrier: cooperation within the dimer. Biochemistry 44:14732–14740
Smith AL (1967) Preparation, properties and conditions for assay of mitochondria, slaughterhouse material, small-scale. Methods Enzymol 10:81–86
Fiore C, Trézéguet V, Le Saux A, Roux P, Schwimmer C, Dianoux A-C, Noël F, Lauquin GJ-M, Brandolin G, Vignais PV (1998) The mitochondrial ADP/ATP carrier: structural, physiological and pathological aspects. Biochimie 80:137–150
Holloway PW (1973) A simple procedure for removal of Triton X-100 from protein samples. Anal Biochem 53:304–308
Pace CN, Vajdos F, Fee L, Grimsley G, Gray T (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci 4:2411–2423
Riccio P, Aquila H, Klingenberg M (1975) Purification of the carboxyatractylate binding protein from mitochondria. FEBS Lett 56:133–138
Le Saux A, Roux P, Trézéguet V, Fiore C, Schwimmer C, Dianoux A-C, Vignais PV, Brandolin G, Lauquin GJ-M (1996) Conformational changes of the yeast mitochondrial adenosine diphosphate/adenosine triphosphate carrier studied through its intrinsic fluorescence. 1. Tryptophanyl residues of the carrier can be mutated without impairing protein activity. Biochemistry 35:16116–16124
Roux P, Le Saux A, Trézéguet V, Fiore C, Schwimmer C, Dianoux AC, Vignais PV, Lauquin GJ-M, Brandolin G (1996) Conformational changes of the yeast mitochondrial adenosine diphosphate/adenosine triphosphate carrier studied through its intrinsic fluorescence. 2. Assignment of tryptophanyl residues of the carrier to the responses to specific ligands. Biochemistry 35:16125–16131
Hatanaka T, Hashimoto M, Majima E, Shinohara Y, Terada H (1999) Functional expression of the tandem-repeated homodimer of the mitochondrial ADP/ATP carrier in Saccharomyces cerevisiae. Biochem Biophys Res Commun 262:726–730
Dassa EP, Dahout-Gonzalez C, Dianoux AC, Brandolin G (2005) Functional characterization and purification of a Saccharomyces cerevisiae ADP/ATP carrier-iso 1 cytochrome c fusion protein. Protein Expr Purif 40:358–369
Marty I, Brandolin G, Gagnon J, Brasseur R, Vignais PV (1992) Topography of the membrane-bound ADP/ATP carrier assessed by enzymatic proteolysis. Biochemistry 31:4058–4065
Pebay-Peyroula E, Dahout-Gonzalez C, Kahn R, Trézéguet V, Lauquin GJ-M, Brandolin G (2003) Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside. Nature 426:39–44
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Arnou, B., Dahout-Gonzalez, C., Pelosi, L., Lauquin, G.JM., Brandolin, G., Trézéguet, V. (2010). Native Membrane Proteins vs. Yeast Recombinant: An Example: The Mitochondrial ADP/ATP Carrier. In: Lacapère, JJ. (eds) Membrane Protein Structure Determination. Methods in Molecular Biology, vol 654. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-762-4_2
Download citation
DOI: https://doi.org/10.1007/978-1-60761-762-4_2
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-761-7
Online ISBN: 978-1-60761-762-4
eBook Packages: Springer Protocols