High phosphatidylinositol 4-phosphate (PI4P)-dependent ATPase activity for the Drs2p-Cdc50p flippase after removal of its N- and C-terminal extensions
| Autor: | Philippe Champeil, Pierre Le Maréchal, Joseph A. Lyons, Poul Nissen, José Luis Vázquez-Ibar, Cédric Montigny, Aurore Jacquot, Thibaud Dieudonné, Jakob Ulstrup, Miriam-Rose Ash, Guillaume Lenoir, Hassina Azouaoui |
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| Přispěvatelé: | Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut des Neurosciences Paris-Saclay (NeuroPSI), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Nordic-EMBL Partnership for Molecular Medicine, and PUMPkin (DANDRITE), Centre for Membrane Pumps in Cells and Disease (PUMPKIN), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Institut des Neurosciences de Paris-Saclay ( Neuro-PSI ), Université Paris-Sud - Paris 11 ( UP11 ) -Centre National de la Recherche Scientifique ( CNRS ), Nordic-EMBL Partnership for Molecular Medicine, and PUMPkin ( DANDRITE ), Centre for Membrane Pumps in Cells and Disease ( PUMPKIN ) |
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Phosphatidylinositol 4-phosphate [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology MESH : Saccharomyces cerevisiae Plasma protein binding Biochemistry MESH: Thrombin chemistry.chemical_compound MESH : Phosphorylation [ SDV.NEU.SC ] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Cognitive Sciences 0302 clinical medicine Adenosine Triphosphate MESH: Saccharomyces cerevisiae Proteins Phosphatidylinositol Phosphates ATP hydrolysis MESH : Arginine lipid-protein interaction MESH: Adenosine Triphosphate MESH : Adenosine Triphosphate Phospholipid Transfer Proteins Phosphorylation Phospholipids MESH : Phospholipid Transfer Proteins medicine.diagnostic_test [SDV.NEU.PC]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Psychology and behavior Hydrolysis Autophosphorylation MESH: Phospholipid Transfer Proteins Thrombin MESH: Arginine [SDV.NEU.SC]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Cognitive Sciences MESH : Protein Binding Phosphatidylserine MESH: Phosphatidylinositol Phosphates MESH: Saccharomyces cerevisiae phosphoinositide [ SDV.NEU.NB ] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology inhibition mechanism MESH: Protein Domains autophosphorylation MESH : Mutation MESH: Hydrolysis Protein Binding CDC50 protein phosphatidylserine Saccharomyces cerevisiae Proteins MESH : Proteolysis MESH: Mutation Proteolysis MESH : Calcium-Transporting ATPases MESH: Proteolysis Calcium-Transporting ATPases Saccharomyces cerevisiae Biology Arginine MESH : Phospholipids MESH: Calcium-Transporting ATPases MESH : Saccharomyces cerevisiae Proteins [ SDV.NEU.PC ] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Psychology and behavior 03 medical and health sciences Protein Domains MESH : Hydrolysis Membrane Biology medicine Journal Article MESH : Phosphatidylinositol Phosphates MESH: Protein Binding Phosphatidylinositol Molecular Biology MESH: Phospholipids MESH: Phosphorylation MESH : Protein Domains MESH : Thrombin Cell Biology Flippase flippase 030104 developmental biology chemistry Mutation 030217 neurology & neurosurgery limited proteolysis |
| Zdroj: | Journal of Biological Chemistry Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2017, 292 (19), pp.7954-7970. ⟨10.1074/jbc.M116.751487⟩ Journal of Biological Chemistry, 2017, 292 (19), pp.7954-7970. ⟨10.1074/jbc.M116.751487⟩ Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2017, 292 (19), pp.7954-7970. 〈10.1074/jbc.M116.751487〉 Azouaoui, H, Montigny, C, Dieudonné, T, Champeil, P, Jacquot, A, Vázquez-Ibar, J L, le Maréchal, P, Ulstrup, J, Ash, M-R, Lyons, J A, Nissen, P & Lenoir, G 2017, ' A High and Phosphatidylinositol-4-phosphate (PI4P)-dependent ATPase Activity for the Drs2p/Cdc50p Flippase after Removal of its N-and C-terminal Extensions ', Journal of Biological Chemistry, vol. 292, pp. 7954-7970 . https://doi.org/10.1074/jbc.M116.751487 |
| ISSN: | 0021-9258 1083-351X |
| Popis: | International audience; 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 Arg104 and after Arg 1290, 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 the trans-Golgi network, followed by full activation by PI4P. |
| Databáze: | OpenAIRE |
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