Abstract
Structural and functional characterization of integral membrane proteins requires milligram amounts of purified sample. Unless the protein you are studying is abundant in native membranes, it will be critical to overexpress the protein of interest in a homologous or heterologous way, and in sufficient quantities for further purification. The situation may become even more complicated if you chose to investigate the structure and function of a complex of two or more membrane proteins. Here, we describe the overexpression of a yeast lipid flippase complex, namely the P4-ATPase Drs2p and its associated subunit Cdc50p, in a coordinated manner. Moreover, we can take advantage of the fact that P4-ATPases, like most other P-type ATPases, form an acid-stable phosphorylated intermediate, to verify that the expressed complex is functional.
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Acknowledgments
We are grateful to Dr. Miriam-Rose Ash for critical reading of the manuscript and for insightful discussions. We also thank Dr. Marc le Maire and Dr. Christine Jaxel for discussions. This work was supported by grants from the Agence Nationale de la Recherche (ANR Blanc program, MIT-2M), the Ile de France region (Domaine d’Intérêt Majeur Maladies Infectieuses, DIM Malinf), and the French Infrastructure for Integrated Structural Biology (FRISBI, ANR-10-INSB-05-01).
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Azouaoui, H., Montigny, C., Jacquot, A., Barry, R., Champeil, P., Lenoir, G. (2016). Coordinated Overexpression in Yeast of a P4-ATPase and Its Associated Cdc50 Subunit: The Case of the Drs2p/Cdc50p Lipid Flippase Complex. In: Bublitz, M. (eds) P-Type ATPases. Methods in Molecular Biology, vol 1377. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3179-8_6
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DOI: https://doi.org/10.1007/978-1-4939-3179-8_6
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3178-1
Online ISBN: 978-1-4939-3179-8
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