title: High phosphatidylinositol 4-phosphate (PI4P)-dependent ATPase activity for the Drs2p-Cdc50p flippase after removal of its N- and C-terminal extensions.
abstract: P4-ATPases, also known as phospholipid flippases, are responsible for creating and maintaining transbilayer lipid asymmetry in eukaryotic cell membranes. Here, we use limited proteolysis to investigate the role of the N and C termini in ATP hydrolysis and auto-inhibition of the yeast flippase Drs2p-Cdc50p. We show that limited proteolysis of the detergent-solubilized and purified yeast flippase may result in more than 1 order of magnitude increase of its ATPase activity, which remains dependent on phosphatidylinositol 4-phosphate (PI4P), a regulator of this lipid flippase, and specific to a phosphatidylserine substrate. Using thrombin as the protease, Cdc50p remains intact and in complex with Drs2p, which is cleaved at two positions, namely after Arg104and after Arg1290, resulting in a homogeneous sample lacking 104 and 65 residues from its N and C termini, respectively. Removal of the 1291-1302-amino acid region of the C-terminal extension is critical for relieving the auto-inhibition of full-length Drs2p, whereas the 1-104 N-terminal residues have an additional but more modest significance for activity. The present results therefore reveal that trimming off appropriate regions of the terminal extensions of Drs2p can greatly increase its ATPase activity in the presence of PI4P and demonstrate that relief of such auto-inhibition remains compatible with subsequent regulation by PI4P. These experiments suggest that activation of the Drs2p-Cdc50p flippase follows a multistep mechanism, with preliminary release of a number of constraints, possibly through the binding of regulatory proteins in thetrans-Golgi network, followed by full activation by PI4P.
authors.affiliation: From the Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, F-91198, Gif-sur-Yvette Cedex, France.
authors.forename: Hassina
authors.initials: H
authors.lastname: Azouaoui
authors.affiliation: From the Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, F-91198, Gif-sur-Yvette Cedex, France.
authors.forename: Cédric
authors.initials: C
authors.lastname: Montigny
authors.affiliation: From the Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, F-91198, Gif-sur-Yvette Cedex, France.
authors.forename: Thibaud
authors.initials: T
authors.lastname: Dieudonné
authors.affiliation: From the Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, F-91198, Gif-sur-Yvette Cedex, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Champeil
authors.affiliation: From the Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, F-91198, Gif-sur-Yvette Cedex, France.
authors.forename: Aurore
authors.initials: A
authors.lastname: Jacquot
authors.affiliation: From the Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, F-91198, Gif-sur-Yvette Cedex, France.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Vázquez-Ibar
authors.affiliation: the Neuro-PSI-UMR CNRS 9197, Bâtiment 430, Université Paris-Sud, Université Paris-Saclay, 91405 Orsay Cedex, France, and.
authors.forename: Pierre
authors.initials: P
authors.lastname: Le Maréchal
authors.affiliation: the DANDRITE, Nordic-EMBL Partnership for Molecular Medicine, and PUMPkin, Department of Molecular Biology and Genetics, Danish National Research Foundation, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark.
authors.forename: Jakob
authors.initials: J
authors.lastname: Ulstrup
authors.affiliation: the DANDRITE, Nordic-EMBL Partnership for Molecular Medicine, and PUMPkin, Department of Molecular Biology and Genetics, Danish National Research Foundation, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark.
authors.forename: Miriam-Rose
authors.identifier: 0000-0002-0573-9854
authors.initials: MR
authors.lastname: Ash
authors.affiliation: the DANDRITE, Nordic-EMBL Partnership for Molecular Medicine, and PUMPkin, Department of Molecular Biology and Genetics, Danish National Research Foundation, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Lyons
authors.affiliation: the DANDRITE, Nordic-EMBL Partnership for Molecular Medicine, and PUMPkin, Department of Molecular Biology and Genetics, Danish National Research Foundation, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark.
authors.forename: Poul
authors.identifier: 0000-0003-0948-6628
authors.initials: P
authors.lastname: Nissen
authors.affiliation: From the Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, F-91198, Gif-sur-Yvette Cedex, France, guillaume.lenoir@i2bc.paris-saclay.fr.
authors.forename: Guillaume
authors.identifier: 0000-0002-8759-5179
authors.initials: G
authors.lastname: Lenoir
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D001120:Arginine / Q000737:chemistry; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D006868:Hydrolysis; D009154:Mutation; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry*; D049294:Phospholipid Transfer Proteins / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D000072417:Protein Domains; D059748:Proteolysis; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013917:Thrombin / Q000737:chemistry
chemical_list: C427246:CDC50 protein, S cerevisiae; C104014:DRS2 protein, S cerevisiae; D018129:Phosphatidylinositol Phosphates; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D029701:Saccharomyces cerevisiae Proteins; C037178:phosphatidylinositol 4-phosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D013917:Thrombin; D000252:Calcium-Transporting ATPases
keywords: CDC50 protein; autophosphorylation; flippase; inhibition mechanism; limited proteolysis; lipid-protein interaction; phosphatidylserine; phosphoinositide