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
(2014) In PLoS ONE 9(11).- 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... (More)
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.
(Less)
- author
- publishing date
- 2014
- type
- Contribution to journal
- publication status
- published
- keywords
- Calcium-Transporting ATPases, Phosphatidylinositol Phosphates, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Journal Article, Research Support, Non-U.S. Gov't
- in
- PLoS ONE
- volume
- 9
- issue
- 11
- article number
- e112176
- publisher
- Public Library of Science (PLoS)
- external identifiers
-
- pmid:25393116
- scopus:84911499372
- ISSN
- 1932-6203
- DOI
- 10.1371/journal.pone.0112176
- language
- English
- LU publication?
- no
- id
- 43440c7e-6459-4f5c-a86a-5338a5f5c4af
- date added to LUP
- 2017-04-29 15:28:49
- date last changed
- 2024-10-14 04:58:17
@article{43440c7e-6459-4f5c-a86a-5338a5f5c4af,
abstract = {{<p>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.</p>}},
author = {{Azouaoui, Hassina and Montigny, Cédric and Ash, Miriam-Rose and Fijalkowski, Frank and Jacquot, Aurore and Grønberg, Christina and López-Marqués, Rosa L and Palmgren, Michael G and Garrigos, Manuel and le Maire, Marc and Decottignies, Paulette and Gourdon, Pontus and Nissen, Poul and Champeil, Philippe and Lenoir, Guillaume}},
issn = {{1932-6203}},
keywords = {{Calcium-Transporting ATPases; Phosphatidylinositol Phosphates; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Journal Article; Research Support, Non-U.S. Gov't}},
language = {{eng}},
number = {{11}},
publisher = {{Public Library of Science (PLoS)}},
series = {{PLoS ONE}},
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}},
url = {{http://dx.doi.org/10.1371/journal.pone.0112176}},
doi = {{10.1371/journal.pone.0112176}},
volume = {{9}},
year = {{2014}},
}