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

Protection of the purified Drs2p-Cdc50p complex from irreversible denaturation.(A) The streptavidin-purified sample (before treatment with Ni2+-TED) was diluted to 17.5 µg/mL into KNG buffer at 30°C, supplemented with 1 mg/mL DDM, in the absence (circles) or presence (triangles) of 0.025 mg/mL POPS, and in the absence (open symbols) or presence (closed symbols) of 0.025 mg/mL PI4P. After various periods of incubation, residual phosphorylation was measured 1 min after addition of [γ-32P]ATP. (B–D) The streptavidin-purified sample (here after treatment with Ni2+-TED), was pre-incubated for various periods at about 150 µg/mL in SSR buffer supplemented with various detergents and lipids at different temperatures, before dilution with two volumes of DDM- and lipid-containing SSR buffer, such that in all cases final conditions included 1 mg/mL DDM, 0.05 mg/mL POPS, and 0.025 mg/mL PI4P. 0.5 µM [γ-32P]ATP was added and the residual ability of the sample to get phosphorylated was measured (after 1.5–2 min at 30°C). Pre-incubation conditions: (B) 20°C, 0.75 mg/mL DDM + 0.025 mg/mL POPS, in the absence (open triangles) or presence (closed triangles) of 0.025 mg/mL PI4P. (C) 20°C, 0.75 mg/mL DDM + 0.75 mg/mL C12E8 + 0.025 mg/mL POPS, in the absence (open squares) or presence (closed squares) of 0.025 mg/mL PI4P. (D) On ice, 0.75 mg/mL DDM + 0.75 mg/mL C12E8 + 0.025 mg/mL POPS in the presence of 0.025 mg/mL PI4P (closed squares), or 0.5 mg/mL DDM + 0.025 mg/mL POPS alone (open triangles).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112176-g008: Protection of the purified Drs2p-Cdc50p complex from irreversible denaturation.(A) The streptavidin-purified sample (before treatment with Ni2+-TED) was diluted to 17.5 µg/mL into KNG buffer at 30°C, supplemented with 1 mg/mL DDM, in the absence (circles) or presence (triangles) of 0.025 mg/mL POPS, and in the absence (open symbols) or presence (closed symbols) of 0.025 mg/mL PI4P. After various periods of incubation, residual phosphorylation was measured 1 min after addition of [γ-32P]ATP. (B–D) The streptavidin-purified sample (here after treatment with Ni2+-TED), was pre-incubated for various periods at about 150 µg/mL in SSR buffer supplemented with various detergents and lipids at different temperatures, before dilution with two volumes of DDM- and lipid-containing SSR buffer, such that in all cases final conditions included 1 mg/mL DDM, 0.05 mg/mL POPS, and 0.025 mg/mL PI4P. 0.5 µM [γ-32P]ATP was added and the residual ability of the sample to get phosphorylated was measured (after 1.5–2 min at 30°C). Pre-incubation conditions: (B) 20°C, 0.75 mg/mL DDM + 0.025 mg/mL POPS, in the absence (open triangles) or presence (closed triangles) of 0.025 mg/mL PI4P. (C) 20°C, 0.75 mg/mL DDM + 0.75 mg/mL C12E8 + 0.025 mg/mL POPS, in the absence (open squares) or presence (closed squares) of 0.025 mg/mL PI4P. (D) On ice, 0.75 mg/mL DDM + 0.75 mg/mL C12E8 + 0.025 mg/mL POPS in the presence of 0.025 mg/mL PI4P (closed squares), or 0.5 mg/mL DDM + 0.025 mg/mL POPS alone (open triangles).

Mentions: When incubation in the presence of DDM took place at 30°C, the Drs2p-Cdc50p complex lost its phosphorylation ability very rapidly (over minutes) in the absence of PS (open circles in Figure 8A). The presence of PS exerted significant, but only partial, protection (Figure 8A). DOPC was even less effective than POPS (data not shown). In contrast, we found that the presence of PI4P afforded complete protection at 30°C, and this was in fact true both in the presence and the absence of POPS (Figure 8A).


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)

Protection of the purified Drs2p-Cdc50p complex from irreversible denaturation.(A) The streptavidin-purified sample (before treatment with Ni2+-TED) was diluted to 17.5 µg/mL into KNG buffer at 30°C, supplemented with 1 mg/mL DDM, in the absence (circles) or presence (triangles) of 0.025 mg/mL POPS, and in the absence (open symbols) or presence (closed symbols) of 0.025 mg/mL PI4P. After various periods of incubation, residual phosphorylation was measured 1 min after addition of [γ-32P]ATP. (B–D) The streptavidin-purified sample (here after treatment with Ni2+-TED), was pre-incubated for various periods at about 150 µg/mL in SSR buffer supplemented with various detergents and lipids at different temperatures, before dilution with two volumes of DDM- and lipid-containing SSR buffer, such that in all cases final conditions included 1 mg/mL DDM, 0.05 mg/mL POPS, and 0.025 mg/mL PI4P. 0.5 µM [γ-32P]ATP was added and the residual ability of the sample to get phosphorylated was measured (after 1.5–2 min at 30°C). Pre-incubation conditions: (B) 20°C, 0.75 mg/mL DDM + 0.025 mg/mL POPS, in the absence (open triangles) or presence (closed triangles) of 0.025 mg/mL PI4P. (C) 20°C, 0.75 mg/mL DDM + 0.75 mg/mL C12E8 + 0.025 mg/mL POPS, in the absence (open squares) or presence (closed squares) of 0.025 mg/mL PI4P. (D) On ice, 0.75 mg/mL DDM + 0.75 mg/mL C12E8 + 0.025 mg/mL POPS in the presence of 0.025 mg/mL PI4P (closed squares), or 0.5 mg/mL DDM + 0.025 mg/mL POPS alone (open triangles).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112176-g008: Protection of the purified Drs2p-Cdc50p complex from irreversible denaturation.(A) The streptavidin-purified sample (before treatment with Ni2+-TED) was diluted to 17.5 µg/mL into KNG buffer at 30°C, supplemented with 1 mg/mL DDM, in the absence (circles) or presence (triangles) of 0.025 mg/mL POPS, and in the absence (open symbols) or presence (closed symbols) of 0.025 mg/mL PI4P. After various periods of incubation, residual phosphorylation was measured 1 min after addition of [γ-32P]ATP. (B–D) The streptavidin-purified sample (here after treatment with Ni2+-TED), was pre-incubated for various periods at about 150 µg/mL in SSR buffer supplemented with various detergents and lipids at different temperatures, before dilution with two volumes of DDM- and lipid-containing SSR buffer, such that in all cases final conditions included 1 mg/mL DDM, 0.05 mg/mL POPS, and 0.025 mg/mL PI4P. 0.5 µM [γ-32P]ATP was added and the residual ability of the sample to get phosphorylated was measured (after 1.5–2 min at 30°C). Pre-incubation conditions: (B) 20°C, 0.75 mg/mL DDM + 0.025 mg/mL POPS, in the absence (open triangles) or presence (closed triangles) of 0.025 mg/mL PI4P. (C) 20°C, 0.75 mg/mL DDM + 0.75 mg/mL C12E8 + 0.025 mg/mL POPS, in the absence (open squares) or presence (closed squares) of 0.025 mg/mL PI4P. (D) On ice, 0.75 mg/mL DDM + 0.75 mg/mL C12E8 + 0.025 mg/mL POPS in the presence of 0.025 mg/mL PI4P (closed squares), or 0.5 mg/mL DDM + 0.025 mg/mL POPS alone (open triangles).
Mentions: When incubation in the presence of DDM took place at 30°C, the Drs2p-Cdc50p complex lost its phosphorylation ability very rapidly (over minutes) in the absence of PS (open circles in Figure 8A). The presence of PS exerted significant, but only partial, protection (Figure 8A). DOPC was even less effective than POPS (data not shown). In contrast, we found that the presence of PI4P afforded complete protection at 30°C, and this was in fact true both in the presence and the absence of POPS (Figure 8A).

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