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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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Co-expression in yeast and functional properties of C- or N-terminally tagged Drs2p and Cdc50p.(A) Functional complementation of the temperature-sensitive phenotype of Δdrs2 and Δcdc50 yeast cells. Yeast cells, either wild-type, Δdrs2, or Δcdc50 mutants, were transformed with plasmids bearing either DRS2 (WT or D560N) tagged with a sequence coding for Bad, or CDC50 tagged with ten histidines (His10). Tags were inserted at the proteins C- or N-termini. Cells transformed with an empty vector (EV) were used as negative control. Serial dilutions of yeast cells were spotted on plates and incubated at the restrictive temperature of 20°C. (B) Co-expression of C-terminally or N-terminally tagged Drs2p and Cdc50p in P3 membranes, as analyzed by western-blotting. For detection using a Biotin or a Histidine probe (top and bottom gels, respectively), 1.5 µg of total protein was loaded per lane and 1 µg total protein was used for detection with an α-Drs2p antibody (intermediate gel). The biotin probe also weakly detected endogenous yeast proteins known to be biotinylated, Acc1p and Pyc1/2p (dashed arrows). Drs2p(t), truncated form of Drs2p; Cdc50p(g), glycosylated forms of Cdc50p [36]; Cdc50p(c), core (unglycosylated) Cdc50p. (C) Phosphorylation from [γ-32P]ATP of P3 membrane fractions co-expressing the tagged Drs2p and Cdc50p proteins. Formation of the phosphoenzyme intermediate was measured in the absence or presence of 1 mM vanadate (open bars and grey bars, respectively). (D) Turnover-dependent dephosphorylation in the presence of DDM and POPS and in the presence (triangles) or absence (circles) of PI4P (see “Materials and Methods”).
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pone-0112176-g001: Co-expression in yeast and functional properties of C- or N-terminally tagged Drs2p and Cdc50p.(A) Functional complementation of the temperature-sensitive phenotype of Δdrs2 and Δcdc50 yeast cells. Yeast cells, either wild-type, Δdrs2, or Δcdc50 mutants, were transformed with plasmids bearing either DRS2 (WT or D560N) tagged with a sequence coding for Bad, or CDC50 tagged with ten histidines (His10). Tags were inserted at the proteins C- or N-termini. Cells transformed with an empty vector (EV) were used as negative control. Serial dilutions of yeast cells were spotted on plates and incubated at the restrictive temperature of 20°C. (B) Co-expression of C-terminally or N-terminally tagged Drs2p and Cdc50p in P3 membranes, as analyzed by western-blotting. For detection using a Biotin or a Histidine probe (top and bottom gels, respectively), 1.5 µg of total protein was loaded per lane and 1 µg total protein was used for detection with an α-Drs2p antibody (intermediate gel). The biotin probe also weakly detected endogenous yeast proteins known to be biotinylated, Acc1p and Pyc1/2p (dashed arrows). Drs2p(t), truncated form of Drs2p; Cdc50p(g), glycosylated forms of Cdc50p [36]; Cdc50p(c), core (unglycosylated) Cdc50p. (C) Phosphorylation from [γ-32P]ATP of P3 membrane fractions co-expressing the tagged Drs2p and Cdc50p proteins. Formation of the phosphoenzyme intermediate was measured in the absence or presence of 1 mM vanadate (open bars and grey bars, respectively). (D) Turnover-dependent dephosphorylation in the presence of DDM and POPS and in the presence (triangles) or absence (circles) of PI4P (see “Materials and Methods”).

Mentions: For experiments with P3 membranes (Figure 1C and 1D), 40-µl samples at 0.5 mg/mL of total protein were pre-incubated on ice in buffer A (100 mM KCl, 5 mM Mg2+ and 50 mM Mops-Tris at pH 7), supplemented with 5 mg/mL DDM in the presence or absence of 0.25 mg/mL POPS or 0.25 mg/mL PI4P. In some cases, 1 mM orthovanadate was added. Phosphorylation was triggered by addition of 0.5 µM [γ-32P]ATP (at 0.25–1 mCi/µmol) on ice (to avoid excessive ATP hydrolysis by Drs2p-unrelated proteins in crude membranes), followed after 25 seconds by acid quenching (typically 1 mL of 500 mM trichloroacetic acid (TCA) + 30 mM H3PO4). Samples were left on ice for more than half an hour after quenching, a period sufficient for aggregation of the precipitated protein and therefore its retention by the filter (this aggregation period was critical in the presence of detergent). This was followed by filtration on either a glass fiber A/E filter or a nitrocellulose GSWP filter, and careful rinsing with dilute acid (50 mM TCA + 3 mM H3PO4). The kinetics of turnover-dependent dephosphorylation were measured by first phosphorylating the sample for 25 seconds on ice under the above conditions and then chasing 32P from the phosphoenzyme by transferring it into a tube pre-equilibrated at 37°C and containing concentrated non-radioactive Mg-ATP (so that its final concentration was 1 mM) for dephosphorylation during the desired time period.


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)

Co-expression in yeast and functional properties of C- or N-terminally tagged Drs2p and Cdc50p.(A) Functional complementation of the temperature-sensitive phenotype of Δdrs2 and Δcdc50 yeast cells. Yeast cells, either wild-type, Δdrs2, or Δcdc50 mutants, were transformed with plasmids bearing either DRS2 (WT or D560N) tagged with a sequence coding for Bad, or CDC50 tagged with ten histidines (His10). Tags were inserted at the proteins C- or N-termini. Cells transformed with an empty vector (EV) were used as negative control. Serial dilutions of yeast cells were spotted on plates and incubated at the restrictive temperature of 20°C. (B) Co-expression of C-terminally or N-terminally tagged Drs2p and Cdc50p in P3 membranes, as analyzed by western-blotting. For detection using a Biotin or a Histidine probe (top and bottom gels, respectively), 1.5 µg of total protein was loaded per lane and 1 µg total protein was used for detection with an α-Drs2p antibody (intermediate gel). The biotin probe also weakly detected endogenous yeast proteins known to be biotinylated, Acc1p and Pyc1/2p (dashed arrows). Drs2p(t), truncated form of Drs2p; Cdc50p(g), glycosylated forms of Cdc50p [36]; Cdc50p(c), core (unglycosylated) Cdc50p. (C) Phosphorylation from [γ-32P]ATP of P3 membrane fractions co-expressing the tagged Drs2p and Cdc50p proteins. Formation of the phosphoenzyme intermediate was measured in the absence or presence of 1 mM vanadate (open bars and grey bars, respectively). (D) Turnover-dependent dephosphorylation in the presence of DDM and POPS and in the presence (triangles) or absence (circles) of PI4P (see “Materials and Methods”).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112176-g001: Co-expression in yeast and functional properties of C- or N-terminally tagged Drs2p and Cdc50p.(A) Functional complementation of the temperature-sensitive phenotype of Δdrs2 and Δcdc50 yeast cells. Yeast cells, either wild-type, Δdrs2, or Δcdc50 mutants, were transformed with plasmids bearing either DRS2 (WT or D560N) tagged with a sequence coding for Bad, or CDC50 tagged with ten histidines (His10). Tags were inserted at the proteins C- or N-termini. Cells transformed with an empty vector (EV) were used as negative control. Serial dilutions of yeast cells were spotted on plates and incubated at the restrictive temperature of 20°C. (B) Co-expression of C-terminally or N-terminally tagged Drs2p and Cdc50p in P3 membranes, as analyzed by western-blotting. For detection using a Biotin or a Histidine probe (top and bottom gels, respectively), 1.5 µg of total protein was loaded per lane and 1 µg total protein was used for detection with an α-Drs2p antibody (intermediate gel). The biotin probe also weakly detected endogenous yeast proteins known to be biotinylated, Acc1p and Pyc1/2p (dashed arrows). Drs2p(t), truncated form of Drs2p; Cdc50p(g), glycosylated forms of Cdc50p [36]; Cdc50p(c), core (unglycosylated) Cdc50p. (C) Phosphorylation from [γ-32P]ATP of P3 membrane fractions co-expressing the tagged Drs2p and Cdc50p proteins. Formation of the phosphoenzyme intermediate was measured in the absence or presence of 1 mM vanadate (open bars and grey bars, respectively). (D) Turnover-dependent dephosphorylation in the presence of DDM and POPS and in the presence (triangles) or absence (circles) of PI4P (see “Materials and Methods”).
Mentions: For experiments with P3 membranes (Figure 1C and 1D), 40-µl samples at 0.5 mg/mL of total protein were pre-incubated on ice in buffer A (100 mM KCl, 5 mM Mg2+ and 50 mM Mops-Tris at pH 7), supplemented with 5 mg/mL DDM in the presence or absence of 0.25 mg/mL POPS or 0.25 mg/mL PI4P. In some cases, 1 mM orthovanadate was added. Phosphorylation was triggered by addition of 0.5 µM [γ-32P]ATP (at 0.25–1 mCi/µmol) on ice (to avoid excessive ATP hydrolysis by Drs2p-unrelated proteins in crude membranes), followed after 25 seconds by acid quenching (typically 1 mL of 500 mM trichloroacetic acid (TCA) + 30 mM H3PO4). Samples were left on ice for more than half an hour after quenching, a period sufficient for aggregation of the precipitated protein and therefore its retention by the filter (this aggregation period was critical in the presence of detergent). This was followed by filtration on either a glass fiber A/E filter or a nitrocellulose GSWP filter, and careful rinsing with dilute acid (50 mM TCA + 3 mM H3PO4). The kinetics of turnover-dependent dephosphorylation were measured by first phosphorylating the sample for 25 seconds on ice under the above conditions and then chasing 32P from the phosphoenzyme by transferring it into a tube pre-equilibrated at 37°C and containing concentrated non-radioactive Mg-ATP (so that its final concentration was 1 mM) for dephosphorylation during the desired time period.

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