A high-yield co-expression system for the purification of an intact drs2p-cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate

Publikation: Bidrag til tidsskriftTidsskriftartikelfagfællebedømt

Standard

A high-yield co-expression system for the purification of an intact drs2p-cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate. / Azouaoui, Hassina; Montigny, Cédric; Ash, Miriam-Rose; Fijalkowski, Franciszek Antoni; Jacquot, Aurore; Grønberg, Christina; Lopez Marques, Rosa Laura; Palmgren, Michael Broberg; Garrigos, Manuel; le Maire, Marc; Decottignies, Paulette; Gourdon, Pontus Emanuel; Nissen, Poul; Champeil, Philippe; Lenoir, Guillaume.

I: PloS one, Bind 9, Nr. 11, e112176, 2014, s. 1-16.

Publikation: Bidrag til tidsskriftTidsskriftartikelfagfællebedømt

Harvard

Azouaoui, H, Montigny, C, Ash, M-R, Fijalkowski, FA, Jacquot, A, Grønberg, C, Lopez Marques, RL, Palmgren, MB, Garrigos, M, le Maire, M, Decottignies, P, Gourdon, PE, Nissen, P, Champeil, P & Lenoir, G 2014, 'A high-yield co-expression system for the purification of an intact drs2p-cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate', PloS one, bind 9, nr. 11, e112176, s. 1-16. https://doi.org/10.1371/journal.pone.0112176

APA

Azouaoui, H., Montigny, C., Ash, M-R., Fijalkowski, F. A., Jacquot, A., Grønberg, C., Lopez Marques, R. L., Palmgren, M. B., Garrigos, M., le Maire, M., Decottignies, P., Gourdon, P. E., Nissen, P., Champeil, P., & Lenoir, G. (2014). A high-yield co-expression system for the purification of an intact drs2p-cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate. PloS one, 9(11), 1-16. [e112176]. https://doi.org/10.1371/journal.pone.0112176

Vancouver

Azouaoui H, Montigny C, Ash M-R, Fijalkowski FA, Jacquot A, Grønberg C o.a. A high-yield co-expression system for the purification of an intact drs2p-cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate. PloS one. 2014;9(11):1-16. e112176. https://doi.org/10.1371/journal.pone.0112176

Author

Azouaoui, Hassina ; Montigny, Cédric ; Ash, Miriam-Rose ; Fijalkowski, Franciszek Antoni ; Jacquot, Aurore ; Grønberg, Christina ; Lopez Marques, Rosa Laura ; Palmgren, Michael Broberg ; Garrigos, Manuel ; le Maire, Marc ; Decottignies, Paulette ; Gourdon, Pontus Emanuel ; Nissen, Poul ; Champeil, Philippe ; Lenoir, Guillaume. / A high-yield co-expression system for the purification of an intact drs2p-cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate. I: PloS one. 2014 ; Bind 9, Nr. 11. s. 1-16.

Bibtex

@article{07db2d431b594019969c8586384a0ba4,
title = "A high-yield co-expression system for the purification of an intact drs2p-cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate",
abstract = "P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic leaflet of plasma membranes, late Golgi membranes, and endosomes. To gain insights into the structure and function of these important membrane pumps, robust protocols for expression and purification are required. In this report, we present a procedure for high-yield co-expression of a yeast flippase, the Drs2p-Cdc50p complex. After recovery of yeast membranes expressing both proteins, efficient purification was achieved in a single step by affinity chromatography on streptavidin beads, yielding ∼1-2 mg purified Drs2p-Cdc50p complex per liter of culture. Importantly, the procedure enabled us to recover a fraction that mainly contained a 1∶1 complex, which was assessed by size-exclusion chromatography and mass spectrometry. The functional properties of the purified complex were examined, including the dependence of its catalytic cycle on specific lipids. The dephosphorylation rate was stimulated in the simultaneous presence of the transported substrate, phosphatidylserine (PS), and the regulatory lipid phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide that plays critical roles in membrane trafficking events from the trans-Golgi network (TGN). Likewise, overall ATP hydrolysis by the complex was critically dependent on the simultaneous presence of PI4P and PS. We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C12E8-induced irreversible inactivation. These results indicate that the Drs2p-Cdc50p complex remains functional after affinity purification and that PI4P as a cofactor tightly controls its stability and catalytic activity. This work offers appealing perspectives for detailed structural and functional characterization of the Drs2p-Cdc50p lipid transport mechanism.",
author = "Hassina Azouaoui and C{\'e}dric Montigny and Miriam-Rose Ash and Fijalkowski, {Franciszek Antoni} and Aurore Jacquot and Christina Gr{\o}nberg and {Lopez Marques}, {Rosa Laura} and Palmgren, {Michael Broberg} and Manuel Garrigos and {le Maire}, Marc and Paulette Decottignies and Gourdon, {Pontus Emanuel} and Poul Nissen and Philippe Champeil and Guillaume Lenoir",
year = "2014",
doi = "10.1371/journal.pone.0112176",
language = "English",
volume = "9",
pages = "1--16",
journal = "PLoS ONE",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "11",

}

RIS

TY - JOUR

T1 - A high-yield co-expression system for the purification of an intact drs2p-cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate

AU - Azouaoui, Hassina

AU - Montigny, Cédric

AU - Ash, Miriam-Rose

AU - Fijalkowski, Franciszek Antoni

AU - Jacquot, Aurore

AU - Grønberg, Christina

AU - Lopez Marques, Rosa Laura

AU - Palmgren, Michael Broberg

AU - Garrigos, Manuel

AU - le Maire, Marc

AU - Decottignies, Paulette

AU - Gourdon, Pontus Emanuel

AU - Nissen, Poul

AU - Champeil, Philippe

AU - Lenoir, Guillaume

PY - 2014

Y1 - 2014

N2 - P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic leaflet of plasma membranes, late Golgi membranes, and endosomes. To gain insights into the structure and function of these important membrane pumps, robust protocols for expression and purification are required. In this report, we present a procedure for high-yield co-expression of a yeast flippase, the Drs2p-Cdc50p complex. After recovery of yeast membranes expressing both proteins, efficient purification was achieved in a single step by affinity chromatography on streptavidin beads, yielding ∼1-2 mg purified Drs2p-Cdc50p complex per liter of culture. Importantly, the procedure enabled us to recover a fraction that mainly contained a 1∶1 complex, which was assessed by size-exclusion chromatography and mass spectrometry. The functional properties of the purified complex were examined, including the dependence of its catalytic cycle on specific lipids. The dephosphorylation rate was stimulated in the simultaneous presence of the transported substrate, phosphatidylserine (PS), and the regulatory lipid phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide that plays critical roles in membrane trafficking events from the trans-Golgi network (TGN). Likewise, overall ATP hydrolysis by the complex was critically dependent on the simultaneous presence of PI4P and PS. We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C12E8-induced irreversible inactivation. These results indicate that the Drs2p-Cdc50p complex remains functional after affinity purification and that PI4P as a cofactor tightly controls its stability and catalytic activity. This work offers appealing perspectives for detailed structural and functional characterization of the Drs2p-Cdc50p lipid transport mechanism.

AB - P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic leaflet of plasma membranes, late Golgi membranes, and endosomes. To gain insights into the structure and function of these important membrane pumps, robust protocols for expression and purification are required. In this report, we present a procedure for high-yield co-expression of a yeast flippase, the Drs2p-Cdc50p complex. After recovery of yeast membranes expressing both proteins, efficient purification was achieved in a single step by affinity chromatography on streptavidin beads, yielding ∼1-2 mg purified Drs2p-Cdc50p complex per liter of culture. Importantly, the procedure enabled us to recover a fraction that mainly contained a 1∶1 complex, which was assessed by size-exclusion chromatography and mass spectrometry. The functional properties of the purified complex were examined, including the dependence of its catalytic cycle on specific lipids. The dephosphorylation rate was stimulated in the simultaneous presence of the transported substrate, phosphatidylserine (PS), and the regulatory lipid phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide that plays critical roles in membrane trafficking events from the trans-Golgi network (TGN). Likewise, overall ATP hydrolysis by the complex was critically dependent on the simultaneous presence of PI4P and PS. We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C12E8-induced irreversible inactivation. These results indicate that the Drs2p-Cdc50p complex remains functional after affinity purification and that PI4P as a cofactor tightly controls its stability and catalytic activity. This work offers appealing perspectives for detailed structural and functional characterization of the Drs2p-Cdc50p lipid transport mechanism.

U2 - 10.1371/journal.pone.0112176

DO - 10.1371/journal.pone.0112176

M3 - Journal article

C2 - 25393116

VL - 9

SP - 1

EP - 16

JO - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 11

M1 - e112176

ER -

ID: 127671205