A High and Phosphatidylinositol-4-phosphate (PI4P)-dependent ATPase Activity for the Drs2p/Cdc50p Flippase after Removal of its N- and C-terminal Extensions

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A High and Phosphatidylinositol-4-phosphate (PI4P)-dependent ATPase Activity for the Drs2p/Cdc50p Flippase after Removal of its N- and C-terminal Extensions. / Azouaoui, Hassina; Montigny, Cedric; Dieudonné, Thibaud; Champeil, Philippe; Jacquot, Aurore; Vázquez-Ibar, José Luis; le Maréchal, Pierre; Ulstrup, Jakob; Ash, Miriam-Rose; Lyons, Joseph A; Nissen, Poul; Lenoir, Guillaume.

I: Journal of Biological Chemistry, Bind 292, 2017, s. 7954-7970.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

Harvard

Azouaoui, H, Montigny, C, Dieudonné, T, Champeil, P, Jacquot, A, Vázquez-Ibar, JL, le Maréchal, P, Ulstrup, J, Ash, M-R, Lyons, JA, 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, bind 292, s. 7954-7970. https://doi.org/10.1074/jbc.M116.751487

APA

Azouaoui, H., Montigny, C., Dieudonné, T., Champeil, P., Jacquot, A., Vázquez-Ibar, J. L., ... 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, 292, 7954-7970. https://doi.org/10.1074/jbc.M116.751487

CBE

Azouaoui H, Montigny C, Dieudonné T, Champeil P, Jacquot A, Vázquez-Ibar JL, le Maréchal P, Ulstrup J, Ash M-R, Lyons JA, 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. 292:7954-7970. https://doi.org/10.1074/jbc.M116.751487

MLA

Vancouver

Azouaoui H, Montigny C, Dieudonné T, Champeil P, Jacquot A, Vázquez-Ibar JL o.a. 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. 2017;292:7954-7970. https://doi.org/10.1074/jbc.M116.751487

Author

Azouaoui, Hassina ; Montigny, Cedric ; Dieudonné, Thibaud ; Champeil, Philippe ; Jacquot, Aurore ; Vázquez-Ibar, José Luis ; le Maréchal, Pierre ; Ulstrup, Jakob ; Ash, Miriam-Rose ; Lyons, Joseph A ; Nissen, Poul ; Lenoir, Guillaume. / A High and Phosphatidylinositol-4-phosphate (PI4P)-dependent ATPase Activity for the Drs2p/Cdc50p Flippase after Removal of its N- and C-terminal Extensions. I: Journal of Biological Chemistry. 2017 ; Bind 292. s. 7954-7970.

Bibtex

@article{abd159bc14e74bea987c872868583ffc,
title = "A High and 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 one 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 R104 and after R1290, resulting in a homogenous sample lacking 104 and 65 residues from its N- and C-termini, respectively. Removal of the 1291-1302 region of the C-terminal extension is critical for relieving the auto-inhibition of full-length Drs2p, while 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 multi-step 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.",
keywords = "Journal Article",
author = "Hassina Azouaoui and Cedric Montigny and Thibaud Dieudonn{\'e} and Philippe Champeil and Aurore Jacquot and V{\'a}zquez-Ibar, {Jos{\'e} Luis} and {le Mar{\'e}chal}, Pierre and Jakob Ulstrup and Miriam-Rose Ash and Lyons, {Joseph A} and Poul Nissen and Guillaume Lenoir",
note = "Copyright {\circledC} 2017, The American Society for Biochemistry and Molecular Biology.",
year = "2017",
doi = "10.1074/jbc.M116.751487",
language = "English",
volume = "292",
pages = "7954--7970",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology, Inc.",

}

RIS

TY - JOUR

T1 - A High and Phosphatidylinositol-4-phosphate (PI4P)-dependent ATPase Activity for the Drs2p/Cdc50p Flippase after Removal of its N- and C-terminal Extensions

AU - Azouaoui, Hassina

AU - Montigny, Cedric

AU - Dieudonné, Thibaud

AU - Champeil, Philippe

AU - Jacquot, Aurore

AU - Vázquez-Ibar, José Luis

AU - le Maréchal, Pierre

AU - Ulstrup, Jakob

AU - Ash, Miriam-Rose

AU - Lyons, Joseph A

AU - Nissen, Poul

AU - Lenoir, Guillaume

N1 - Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

PY - 2017

Y1 - 2017

N2 - 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 one 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 R104 and after R1290, resulting in a homogenous sample lacking 104 and 65 residues from its N- and C-termini, respectively. Removal of the 1291-1302 region of the C-terminal extension is critical for relieving the auto-inhibition of full-length Drs2p, while 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 multi-step 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.

AB - 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 one 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 R104 and after R1290, resulting in a homogenous sample lacking 104 and 65 residues from its N- and C-termini, respectively. Removal of the 1291-1302 region of the C-terminal extension is critical for relieving the auto-inhibition of full-length Drs2p, while 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 multi-step 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.

KW - Journal Article

U2 - 10.1074/jbc.M116.751487

DO - 10.1074/jbc.M116.751487

M3 - Journal article

VL - 292

SP - 7954

EP - 7970

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

ER -