Limits...
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.

Show MeSH

Related in: MedlinePlus

Phosphorylation and dephosphorylation properties of the purified Drs2p-Cdc50p complex.(A) Time course of ATP-dependent phosphorylation of the purified Drs2p-Cdc50p complex at various concentrations of [γ-32P]ATP. The streptavidin-purified and Ni2+-TED-treated sample, diluted to about 50 µg/mL in SSR on ice in the presence of 1 mg/mL DDM and 0.05 mg/mL POPS, was supplemented with [γ-32P]ATP (typically 4 µL of concentrated ATP was added to 40 µL enzyme) and brought to 30°C for various periods before acid quenching and filtration. (B) ATP-dependence of the maximal phosphorylation level, in the absence (open circles) or presence (open triangles) of PI4P at 0.025 mg/mL. In some cases, the purified sample had been first pre-incubated with 1 mM vanadate (grey symbols). (C and D) The streptavidin-purified sample (here before Ni2+-TED treatment, and diluted to about 60 µg/mL) was supplemented with 0.5 µM [γ-32P]ATP in the absence (C) or presence (D) of PI4P, and phosphorylation at 30°C was measured (white symbols). Grey symbols refer to experiments performed in the presence of vanadate. To trigger ATP-induced chase of the radioactively-labelled phosphoenzyme, 1 mM non-radioactive Mg-ATP was added after 1.5 minute (black symbols, in the absence of vanadate).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230938&req=5

pone-0112176-g005: Phosphorylation and dephosphorylation properties of the purified Drs2p-Cdc50p complex.(A) Time course of ATP-dependent phosphorylation of the purified Drs2p-Cdc50p complex at various concentrations of [γ-32P]ATP. The streptavidin-purified and Ni2+-TED-treated sample, diluted to about 50 µg/mL in SSR on ice in the presence of 1 mg/mL DDM and 0.05 mg/mL POPS, was supplemented with [γ-32P]ATP (typically 4 µL of concentrated ATP was added to 40 µL enzyme) and brought to 30°C for various periods before acid quenching and filtration. (B) ATP-dependence of the maximal phosphorylation level, in the absence (open circles) or presence (open triangles) of PI4P at 0.025 mg/mL. In some cases, the purified sample had been first pre-incubated with 1 mM vanadate (grey symbols). (C and D) The streptavidin-purified sample (here before Ni2+-TED treatment, and diluted to about 60 µg/mL) was supplemented with 0.5 µM [γ-32P]ATP in the absence (C) or presence (D) of PI4P, and phosphorylation at 30°C was measured (white symbols). Grey symbols refer to experiments performed in the presence of vanadate. To trigger ATP-induced chase of the radioactively-labelled phosphoenzyme, 1 mM non-radioactive Mg-ATP was added after 1.5 minute (black symbols, in the absence of vanadate).

Mentions: Firstly, the purified Drs2p-Cdc50p complex is efficiently phosphorylated from [γ-32P]ATP, up to a forty-fold higher level than solubilized crude membranes (about 1.6 nmol/mg in Figure 5A, compared with 0.04 nmol/mg in Figure 1D). We also found that the observed phosphorylation level was already nearly saturated at a Mg-ATP concentration of 0.5 µM (Figure 5A). This sub-micromolar apparent affinity for Mg-ATP showed up both in the absence and presence of PI4P, whereas in the presence of vanadate the apparent affinity for ATP was much lower (Figure 5B).


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)

Phosphorylation and dephosphorylation properties of the purified Drs2p-Cdc50p complex.(A) Time course of ATP-dependent phosphorylation of the purified Drs2p-Cdc50p complex at various concentrations of [γ-32P]ATP. The streptavidin-purified and Ni2+-TED-treated sample, diluted to about 50 µg/mL in SSR on ice in the presence of 1 mg/mL DDM and 0.05 mg/mL POPS, was supplemented with [γ-32P]ATP (typically 4 µL of concentrated ATP was added to 40 µL enzyme) and brought to 30°C for various periods before acid quenching and filtration. (B) ATP-dependence of the maximal phosphorylation level, in the absence (open circles) or presence (open triangles) of PI4P at 0.025 mg/mL. In some cases, the purified sample had been first pre-incubated with 1 mM vanadate (grey symbols). (C and D) The streptavidin-purified sample (here before Ni2+-TED treatment, and diluted to about 60 µg/mL) was supplemented with 0.5 µM [γ-32P]ATP in the absence (C) or presence (D) of PI4P, and phosphorylation at 30°C was measured (white symbols). Grey symbols refer to experiments performed in the presence of vanadate. To trigger ATP-induced chase of the radioactively-labelled phosphoenzyme, 1 mM non-radioactive Mg-ATP was added after 1.5 minute (black symbols, in the absence of vanadate).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112176-g005: Phosphorylation and dephosphorylation properties of the purified Drs2p-Cdc50p complex.(A) Time course of ATP-dependent phosphorylation of the purified Drs2p-Cdc50p complex at various concentrations of [γ-32P]ATP. The streptavidin-purified and Ni2+-TED-treated sample, diluted to about 50 µg/mL in SSR on ice in the presence of 1 mg/mL DDM and 0.05 mg/mL POPS, was supplemented with [γ-32P]ATP (typically 4 µL of concentrated ATP was added to 40 µL enzyme) and brought to 30°C for various periods before acid quenching and filtration. (B) ATP-dependence of the maximal phosphorylation level, in the absence (open circles) or presence (open triangles) of PI4P at 0.025 mg/mL. In some cases, the purified sample had been first pre-incubated with 1 mM vanadate (grey symbols). (C and D) The streptavidin-purified sample (here before Ni2+-TED treatment, and diluted to about 60 µg/mL) was supplemented with 0.5 µM [γ-32P]ATP in the absence (C) or presence (D) of PI4P, and phosphorylation at 30°C was measured (white symbols). Grey symbols refer to experiments performed in the presence of vanadate. To trigger ATP-induced chase of the radioactively-labelled phosphoenzyme, 1 mM non-radioactive Mg-ATP was added after 1.5 minute (black symbols, in the absence of vanadate).
Mentions: Firstly, the purified Drs2p-Cdc50p complex is efficiently phosphorylated from [γ-32P]ATP, up to a forty-fold higher level than solubilized crude membranes (about 1.6 nmol/mg in Figure 5A, compared with 0.04 nmol/mg in Figure 1D). We also found that the observed phosphorylation level was already nearly saturated at a Mg-ATP concentration of 0.5 µM (Figure 5A). This sub-micromolar apparent affinity for Mg-ATP showed up both in the absence and presence of PI4P, whereas in the presence of vanadate the apparent affinity for ATP was much lower (Figure 5B).

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