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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 H, Montigny C, Ash MR, Fijalkowski F, Jacquot A, Grønberg C, López-Marqués RL, Palmgren MG, Garrigos M, le Maire M, Decottignies P, Gourdon P, Nissen P, Champeil P, Lenoir G - PLoS ONE (2014)

Bottom Line: 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.This work offers appealing perspectives for detailed structural and functional characterization of the Drs2p-Cdc50p lipid transport mechanism.

View Article: PubMed Central - PubMed

Affiliation: Univ Paris-Sud, UMR 8221, Orsay, France; CEA, iBiTec-S (Institut de Biologie et de Technologies de Saclay), SB2SM (Service de Bioénergétique, Biologie Structurale et Mécanismes), Laboratoire des Protéines Membranaires, Gif-sur-Yvette, France; CNRS, UMR 8221, Gif-sur-Yvette, France.

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.

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Related in: MedlinePlus

The vast majority of Drs2p is in complex with Cdc50p, in a 1/1 stoichiometry.(A) Yeasts were transformed either with a regular Bad-Drs2p/His10-Cdc50p construct (with N-terminal tags) or with a related construct in which the TEV cleavage site between the His10 tag and Cdc50p had been omitted. This resulted, after streptavidin-based purification, in either the classical sample (D–C) or in a sample where Cdc50p remains tagged with His10 (D-His10C). For both samples, the streptavidin-eluted fractions were diluted 5-fold (to about 70 µg/mL) in KNG buffer supplemented with 1 mg/mL DDM, 0.025 mg/mL PS and 0.025 mg/mL PI4P, and 300 µL of each diluted sample was mixed with 5 mg of dry Ni2+-TED resin (previously washed with the dilution buffer) and incubated on a wheel for 45 minutes in the cold room. Initial diluted samples (Es), and unbound material (FT), were loaded onto a 10% SDS-PAGE and stained with silver nitrate. For D-His10C, the Es sample was further diluted 3-fold and 10-fold and aliquots (Es/10 and Es/3) were loaded for comparison with the FT sample. (B) 2.5 µg and 0.5 µg of streptavidin-purified Drs2p-Cdc50p complex were loaded onto a haloalkane-containing 4–20% gradient gel for both in-gel fluorescence analysis (left, Fluo) and subsequent Coomassie Blue staining of the same gel (right, CB).
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pone-0112176-g003: The vast majority of Drs2p is in complex with Cdc50p, in a 1/1 stoichiometry.(A) Yeasts were transformed either with a regular Bad-Drs2p/His10-Cdc50p construct (with N-terminal tags) or with a related construct in which the TEV cleavage site between the His10 tag and Cdc50p had been omitted. This resulted, after streptavidin-based purification, in either the classical sample (D–C) or in a sample where Cdc50p remains tagged with His10 (D-His10C). For both samples, the streptavidin-eluted fractions were diluted 5-fold (to about 70 µg/mL) in KNG buffer supplemented with 1 mg/mL DDM, 0.025 mg/mL PS and 0.025 mg/mL PI4P, and 300 µL of each diluted sample was mixed with 5 mg of dry Ni2+-TED resin (previously washed with the dilution buffer) and incubated on a wheel for 45 minutes in the cold room. Initial diluted samples (Es), and unbound material (FT), were loaded onto a 10% SDS-PAGE and stained with silver nitrate. For D-His10C, the Es sample was further diluted 3-fold and 10-fold and aliquots (Es/10 and Es/3) were loaded for comparison with the FT sample. (B) 2.5 µg and 0.5 µg of streptavidin-purified Drs2p-Cdc50p complex were loaded onto a haloalkane-containing 4–20% gradient gel for both in-gel fluorescence analysis (left, Fluo) and subsequent Coomassie Blue staining of the same gel (right, CB).

Mentions: Final removal of the His6-tagged TEV protease was easily done by incubating the streptavidin-purified eluate (Es) with a nickel affinity resin, with only little non-specific loss of Drs2p and Cdc50p (Figure 3A).


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 H, Montigny C, Ash MR, Fijalkowski F, Jacquot A, Grønberg C, López-Marqués RL, Palmgren MG, Garrigos M, le Maire M, Decottignies P, Gourdon P, Nissen P, Champeil P, Lenoir G - PLoS ONE (2014)

The vast majority of Drs2p is in complex with Cdc50p, in a 1/1 stoichiometry.(A) Yeasts were transformed either with a regular Bad-Drs2p/His10-Cdc50p construct (with N-terminal tags) or with a related construct in which the TEV cleavage site between the His10 tag and Cdc50p had been omitted. This resulted, after streptavidin-based purification, in either the classical sample (D–C) or in a sample where Cdc50p remains tagged with His10 (D-His10C). For both samples, the streptavidin-eluted fractions were diluted 5-fold (to about 70 µg/mL) in KNG buffer supplemented with 1 mg/mL DDM, 0.025 mg/mL PS and 0.025 mg/mL PI4P, and 300 µL of each diluted sample was mixed with 5 mg of dry Ni2+-TED resin (previously washed with the dilution buffer) and incubated on a wheel for 45 minutes in the cold room. Initial diluted samples (Es), and unbound material (FT), were loaded onto a 10% SDS-PAGE and stained with silver nitrate. For D-His10C, the Es sample was further diluted 3-fold and 10-fold and aliquots (Es/10 and Es/3) were loaded for comparison with the FT sample. (B) 2.5 µg and 0.5 µg of streptavidin-purified Drs2p-Cdc50p complex were loaded onto a haloalkane-containing 4–20% gradient gel for both in-gel fluorescence analysis (left, Fluo) and subsequent Coomassie Blue staining of the same gel (right, CB).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4230938&req=5

pone-0112176-g003: The vast majority of Drs2p is in complex with Cdc50p, in a 1/1 stoichiometry.(A) Yeasts were transformed either with a regular Bad-Drs2p/His10-Cdc50p construct (with N-terminal tags) or with a related construct in which the TEV cleavage site between the His10 tag and Cdc50p had been omitted. This resulted, after streptavidin-based purification, in either the classical sample (D–C) or in a sample where Cdc50p remains tagged with His10 (D-His10C). For both samples, the streptavidin-eluted fractions were diluted 5-fold (to about 70 µg/mL) in KNG buffer supplemented with 1 mg/mL DDM, 0.025 mg/mL PS and 0.025 mg/mL PI4P, and 300 µL of each diluted sample was mixed with 5 mg of dry Ni2+-TED resin (previously washed with the dilution buffer) and incubated on a wheel for 45 minutes in the cold room. Initial diluted samples (Es), and unbound material (FT), were loaded onto a 10% SDS-PAGE and stained with silver nitrate. For D-His10C, the Es sample was further diluted 3-fold and 10-fold and aliquots (Es/10 and Es/3) were loaded for comparison with the FT sample. (B) 2.5 µg and 0.5 µg of streptavidin-purified Drs2p-Cdc50p complex were loaded onto a haloalkane-containing 4–20% gradient gel for both in-gel fluorescence analysis (left, Fluo) and subsequent Coomassie Blue staining of the same gel (right, CB).
Mentions: Final removal of the His6-tagged TEV protease was easily done by incubating the streptavidin-purified eluate (Es) with a nickel affinity resin, with only little non-specific loss of Drs2p and Cdc50p (Figure 3A).

Bottom Line: 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.This work offers appealing perspectives for detailed structural and functional characterization of the Drs2p-Cdc50p lipid transport mechanism.

View Article: PubMed Central - PubMed

Affiliation: Univ Paris-Sud, UMR 8221, Orsay, France; CEA, iBiTec-S (Institut de Biologie et de Technologies de Saclay), SB2SM (Service de Bioénergétique, Biologie Structurale et Mécanismes), Laboratoire des Protéines Membranaires, Gif-sur-Yvette, France; CNRS, UMR 8221, Gif-sur-Yvette, France.

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.

Show MeSH
Related in: MedlinePlus