Coordinated Overexpression in Yeast of a P4-ATPase and Its Associated Cdc50 Subunit: The Case of the Drs2p/Cdc50p Lipid Flippase Complex.

Hassina Azouaoui (1) , Cédric Montigny (1) , Aurore Jacquot (1, 2) , Raphaëlle Barry (1, 3) , Philippe Champeil (1) , Guillaume Lenoir (1)

1 I2BC - Institut de Biologie Intégrative de la Cellule
2 BPMP - Biochimie et Physiologie Moléculaire des Plantes
3 Institut de la Vision

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.

Mots clés: Membrane protein; P4-ATPase; Cdc50 protein; Co-expression; Yeast; Saccharomyces cerevisiae; Lipid transport; Phosphorylation

Mots clés: levure; saccharomyces cerevisiae; phosphorilation; transport de lipides; atpase

Domaines: Biologie végétale

Hassina Azouaoui, Cédric Montigny, Aurore Jacquot, Raphaëlle Barry, Philippe Champeil, et al.. Coordinated Overexpression in Yeast of a P4-ATPase and Its Associated Cdc50 Subunit: The Case of the Drs2p/Cdc50p Lipid Flippase Complex.. Methods in Molecular Biology, 2015, Methods in Molecular Biology, 1377 (Chapter 6), pp.37-55. ⟨10.1007/978-1-4939-3179-8_6⟩. ⟨hal-01354261⟩