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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

Lipid-dependence and Mg-ATP-dependence of the ATPase activity of the purified sample.(A) The ATPase activity of the streptavidin-purified and Ni2+-TED-treated sample, diluted to about 50 µg/mL, was measured at 30°C in SSR buffer supplemented with 1 mM Mg-ATP and 1 mg/mL DDM, as well as with PI4P at various concentrations in the additional presence of 0.05 mg/mL POPS (circles), or with POPS at various concentrations in the additional presence of 0.025 mg/mL PI4P (triangles). (B) The ATPase activity of a sample purified in the absence of PS (and diluted to about 100 µg/mL) was measured at 30°C in KNG buffer supplemented with 1 mg/mL DDM, 1 mM Mg-ATP, and various lipids (all at 0.05 mg/mL), in the absence (hatched bars) or presence (open bars) of 0.025 mg/mL PI4P. Inset: Coomassie Blue staining of this purified sample after SDS-PAGE. (C) The same purified sample as the one for Panel A was used, but in this case the activity was measured at various concentrations of Mg-ATP, in the additional presence of an ATP-regenerating system (and in the presence of 1 mg/mL DDM, 0.05 mg/mL POPS and 0.025 mg/mL PI4P). (D) Plot of the results of the experiment displayed in (C) for determination of the apparent affinity for ATP, in the presence (open triangles) or absence (open circles) of PI4P. [Mg-ATP]1/2 is the concentration of ATP necessary to reach half of the maximal velocity. (E and F) After a 1-hour pre-incubation period on ice either with various VOx concentrations (E) or with various BeF3− or AlF4− concentrations (F), the ATPase activity of Drs2p-Cdc50p was measured at 30°C in SSR buffer supplemented with 1 mg/mL DDM, 0.05 mg/mL POPS, and 1 mM ATP, in the absence (grey bars) or presence (open bars), of 0.025 mg/mL PI4P.
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pone-0112176-g007: Lipid-dependence and Mg-ATP-dependence of the ATPase activity of the purified sample.(A) The ATPase activity of the streptavidin-purified and Ni2+-TED-treated sample, diluted to about 50 µg/mL, was measured at 30°C in SSR buffer supplemented with 1 mM Mg-ATP and 1 mg/mL DDM, as well as with PI4P at various concentrations in the additional presence of 0.05 mg/mL POPS (circles), or with POPS at various concentrations in the additional presence of 0.025 mg/mL PI4P (triangles). (B) The ATPase activity of a sample purified in the absence of PS (and diluted to about 100 µg/mL) was measured at 30°C in KNG buffer supplemented with 1 mg/mL DDM, 1 mM Mg-ATP, and various lipids (all at 0.05 mg/mL), in the absence (hatched bars) or presence (open bars) of 0.025 mg/mL PI4P. Inset: Coomassie Blue staining of this purified sample after SDS-PAGE. (C) The same purified sample as the one for Panel A was used, but in this case the activity was measured at various concentrations of Mg-ATP, in the additional presence of an ATP-regenerating system (and in the presence of 1 mg/mL DDM, 0.05 mg/mL POPS and 0.025 mg/mL PI4P). (D) Plot of the results of the experiment displayed in (C) for determination of the apparent affinity for ATP, in the presence (open triangles) or absence (open circles) of PI4P. [Mg-ATP]1/2 is the concentration of ATP necessary to reach half of the maximal velocity. (E and F) After a 1-hour pre-incubation period on ice either with various VOx concentrations (E) or with various BeF3− or AlF4− concentrations (F), the ATPase activity of Drs2p-Cdc50p was measured at 30°C in SSR buffer supplemented with 1 mg/mL DDM, 0.05 mg/mL POPS, and 1 mM ATP, in the absence (grey bars) or presence (open bars), of 0.025 mg/mL PI4P.

Mentions: Now focusing in more detail on the PI4P-dependent ATPase activity of WT Drs2p-Cdc50p complex, this activity appeared to have a number of very attractive features. In the presence of 1 mg/mL DDM and 0.05 mg/mL POPS, it was stimulated by PI4P to a high degree and the apparent [PI4P]1/2 was of the order of a few µg/mL (or a few µM) only (Figure 7A) (since PI4P does not distribute equally in the total volume but is mainly found in mixed micelles together with DDM, here used at 1 mg/mL, this apparent affinity is more significantly expressed in terms of PI4P/DDM ratio: [PI4P]1/2 was of the order of a few µg of PI4P per mg of DDM). Compared with this high affinity, the apparent affinity for PS (Figure 7A), in the presence of a nearly saturating concentration of PI4P (0.025 mg/mL), was poorer: [PS]1/2 was certainly higher than 20 µg/mL. Note that since purification was performed in the presence of PS, the assay medium contained in all cases ∼80 µg/mL of residual PS derived from the purification procedure, hence the uncertainty about the activity in the total absence of PS.


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)

Lipid-dependence and Mg-ATP-dependence of the ATPase activity of the purified sample.(A) The ATPase activity of the streptavidin-purified and Ni2+-TED-treated sample, diluted to about 50 µg/mL, was measured at 30°C in SSR buffer supplemented with 1 mM Mg-ATP and 1 mg/mL DDM, as well as with PI4P at various concentrations in the additional presence of 0.05 mg/mL POPS (circles), or with POPS at various concentrations in the additional presence of 0.025 mg/mL PI4P (triangles). (B) The ATPase activity of a sample purified in the absence of PS (and diluted to about 100 µg/mL) was measured at 30°C in KNG buffer supplemented with 1 mg/mL DDM, 1 mM Mg-ATP, and various lipids (all at 0.05 mg/mL), in the absence (hatched bars) or presence (open bars) of 0.025 mg/mL PI4P. Inset: Coomassie Blue staining of this purified sample after SDS-PAGE. (C) The same purified sample as the one for Panel A was used, but in this case the activity was measured at various concentrations of Mg-ATP, in the additional presence of an ATP-regenerating system (and in the presence of 1 mg/mL DDM, 0.05 mg/mL POPS and 0.025 mg/mL PI4P). (D) Plot of the results of the experiment displayed in (C) for determination of the apparent affinity for ATP, in the presence (open triangles) or absence (open circles) of PI4P. [Mg-ATP]1/2 is the concentration of ATP necessary to reach half of the maximal velocity. (E and F) After a 1-hour pre-incubation period on ice either with various VOx concentrations (E) or with various BeF3− or AlF4− concentrations (F), the ATPase activity of Drs2p-Cdc50p was measured at 30°C in SSR buffer supplemented with 1 mg/mL DDM, 0.05 mg/mL POPS, and 1 mM ATP, in the absence (grey bars) or presence (open bars), of 0.025 mg/mL PI4P.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112176-g007: Lipid-dependence and Mg-ATP-dependence of the ATPase activity of the purified sample.(A) The ATPase activity of the streptavidin-purified and Ni2+-TED-treated sample, diluted to about 50 µg/mL, was measured at 30°C in SSR buffer supplemented with 1 mM Mg-ATP and 1 mg/mL DDM, as well as with PI4P at various concentrations in the additional presence of 0.05 mg/mL POPS (circles), or with POPS at various concentrations in the additional presence of 0.025 mg/mL PI4P (triangles). (B) The ATPase activity of a sample purified in the absence of PS (and diluted to about 100 µg/mL) was measured at 30°C in KNG buffer supplemented with 1 mg/mL DDM, 1 mM Mg-ATP, and various lipids (all at 0.05 mg/mL), in the absence (hatched bars) or presence (open bars) of 0.025 mg/mL PI4P. Inset: Coomassie Blue staining of this purified sample after SDS-PAGE. (C) The same purified sample as the one for Panel A was used, but in this case the activity was measured at various concentrations of Mg-ATP, in the additional presence of an ATP-regenerating system (and in the presence of 1 mg/mL DDM, 0.05 mg/mL POPS and 0.025 mg/mL PI4P). (D) Plot of the results of the experiment displayed in (C) for determination of the apparent affinity for ATP, in the presence (open triangles) or absence (open circles) of PI4P. [Mg-ATP]1/2 is the concentration of ATP necessary to reach half of the maximal velocity. (E and F) After a 1-hour pre-incubation period on ice either with various VOx concentrations (E) or with various BeF3− or AlF4− concentrations (F), the ATPase activity of Drs2p-Cdc50p was measured at 30°C in SSR buffer supplemented with 1 mg/mL DDM, 0.05 mg/mL POPS, and 1 mM ATP, in the absence (grey bars) or presence (open bars), of 0.025 mg/mL PI4P.
Mentions: Now focusing in more detail on the PI4P-dependent ATPase activity of WT Drs2p-Cdc50p complex, this activity appeared to have a number of very attractive features. In the presence of 1 mg/mL DDM and 0.05 mg/mL POPS, it was stimulated by PI4P to a high degree and the apparent [PI4P]1/2 was of the order of a few µg/mL (or a few µM) only (Figure 7A) (since PI4P does not distribute equally in the total volume but is mainly found in mixed micelles together with DDM, here used at 1 mg/mL, this apparent affinity is more significantly expressed in terms of PI4P/DDM ratio: [PI4P]1/2 was of the order of a few µg of PI4P per mg of DDM). Compared with this high affinity, the apparent affinity for PS (Figure 7A), in the presence of a nearly saturating concentration of PI4P (0.025 mg/mL), was poorer: [PS]1/2 was certainly higher than 20 µg/mL. Note that since purification was performed in the presence of PS, the assay medium contained in all cases ∼80 µg/mL of residual PS derived from the purification procedure, hence the uncertainty about the activity in the total absence of PS.

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