title: Coordinated Overexpression in Yeast of a P4-ATPase and Its Associated Cdc50 Subunit: The Case of the Drs2p/Cdc50p Lipid Flippase Complex.
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.
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC) - UMR 9198 CEA/CNRS/Université Paris-Sud, Gif-sur-Yvette Cedex, 91191, France.|CEA, iBiTec-S/SB2SM, CEA Saclay, Gif-sur-Yvette, 91191, France.
authors.forename: Hassina
authors.initials: H
authors.lastname: Azouaoui
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC) - UMR 9198 CEA/CNRS/Université Paris-Sud, Gif-sur-Yvette Cedex, 91191, France.|CEA, iBiTec-S/SB2SM, CEA Saclay, Gif-sur-Yvette, 91191, France.
authors.forename: Cédric
authors.initials: C
authors.lastname: Montigny
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC) - UMR 9198 CEA/CNRS/Université Paris-Sud, Gif-sur-Yvette Cedex, 91191, France.|CEA, iBiTec-S/SB2SM, CEA Saclay, Gif-sur-Yvette, 91191, France.|UMR 5004, Biochimie et Physiologie Moléculaire des Plantes, Institut de Biologie Intégrative des Plantes-Claude Grignon, Agro-M/CNRS/INRA/SupAgro/UM2, Montpellier, 34060, France.
authors.forename: Aurore
authors.initials: A
authors.lastname: Jacquot
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC) - UMR 9198 CEA/CNRS/Université Paris-Sud, Gif-sur-Yvette Cedex, 91191, France.|CEA, iBiTec-S/SB2SM, CEA Saclay, Gif-sur-Yvette, 91191, France.|INSERM, U968, Paris, 75012, France.|UMR_S 968, Institut de la Vision, Sorbonne Universités, UPMC Univ Paris 06, Paris, 75012, France.|CNRS, UMR 7210, Paris, 75012, France.
authors.forename: Raphaëlle
authors.initials: R
authors.lastname: Barry
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC) - UMR 9198 CEA/CNRS/Université Paris-Sud, Gif-sur-Yvette Cedex, 91191, France.|CEA, iBiTec-S/SB2SM, CEA Saclay, Gif-sur-Yvette, 91191, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Champeil
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC) - UMR 9198 CEA/CNRS/Université Paris-Sud, Gif-sur-Yvette Cedex, 91191, France. guillaume.lenoir@cea.fr.|CEA, iBiTec-S/SB2SM, CEA Saclay, Gif-sur-Yvette, 91191, France. guillaume.lenoir@cea.fr.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Lenoir
mesh_terms: D000252:Calcium-Transporting ATPases / Q000096:biosynthesis* / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry; D046912:Multiprotein Complexes / Q000737:chemistry; D010743:Phospholipids; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: C427246:CDC50 protein, S cerevisiae; C104014:DRS2 protein, S cerevisiae; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D010743:Phospholipids; D029701:Saccharomyces cerevisiae Proteins; D000252:Calcium-Transporting ATPases
keywords: Cdc50 protein; Co-expression; Lipid transport; Membrane protein; P4-ATPase; Phosphorylation; Saccharomyces cerevisiae; Yeast