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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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ATPase activities of various streptavidin-purified samples.(A) Coomassie Blue staining after SDS-PAGE of purified wild-type complex (DWT-C), D560N variant (DD560N-C), E342Q variant (DE342Q-C), and wild-type Drs2p expressed alone. (B–C and E–F) The ATPase activity of the same samples (after 5-fold dilution resulting in about 60 µg/mL Drs2p in the case of the WT enzyme) was measured at 30°C in a KNG medium supplemented with 1 mg/mL DDM, 0.025 mg/mL PS and 1 mM Mg-ATP, in the absence (circles and dashed lines) or presence (triangles and continuous lines) of 0.025 mg/mL PI4P. The dotted line in panels C-F is given for easier comparison with results for WT. (D) Functional complementation of the temperature-sensitive phenotype of Δdrs2 yeast cells. Yeast cells, either wild-type or Δdrs2, were transformed with plasmids bearing DRS2 tagged at its 5′ end, either WT or E342Q. 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.
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pone-0112176-g006: ATPase activities of various streptavidin-purified samples.(A) Coomassie Blue staining after SDS-PAGE of purified wild-type complex (DWT-C), D560N variant (DD560N-C), E342Q variant (DE342Q-C), and wild-type Drs2p expressed alone. (B–C and E–F) The ATPase activity of the same samples (after 5-fold dilution resulting in about 60 µg/mL Drs2p in the case of the WT enzyme) was measured at 30°C in a KNG medium supplemented with 1 mg/mL DDM, 0.025 mg/mL PS and 1 mM Mg-ATP, in the absence (circles and dashed lines) or presence (triangles and continuous lines) of 0.025 mg/mL PI4P. The dotted line in panels C-F is given for easier comparison with results for WT. (D) Functional complementation of the temperature-sensitive phenotype of Δdrs2 yeast cells. Yeast cells, either wild-type or Δdrs2, were transformed with plasmids bearing DRS2 tagged at its 5′ end, either WT or E342Q. 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.

Mentions: As most of the ATP hydrolyzing contaminants in yeast membranes have presumably been removed during purification, the ATPase activity of the Drs2p-Cdc50p complex could be examined. At 30°C, we were indeed able to detect ATPase activity, and also to reveal, for the WT enzyme, its strong dependence on PI4P (Figure 6B).


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)

ATPase activities of various streptavidin-purified samples.(A) Coomassie Blue staining after SDS-PAGE of purified wild-type complex (DWT-C), D560N variant (DD560N-C), E342Q variant (DE342Q-C), and wild-type Drs2p expressed alone. (B–C and E–F) The ATPase activity of the same samples (after 5-fold dilution resulting in about 60 µg/mL Drs2p in the case of the WT enzyme) was measured at 30°C in a KNG medium supplemented with 1 mg/mL DDM, 0.025 mg/mL PS and 1 mM Mg-ATP, in the absence (circles and dashed lines) or presence (triangles and continuous lines) of 0.025 mg/mL PI4P. The dotted line in panels C-F is given for easier comparison with results for WT. (D) Functional complementation of the temperature-sensitive phenotype of Δdrs2 yeast cells. Yeast cells, either wild-type or Δdrs2, were transformed with plasmids bearing DRS2 tagged at its 5′ end, either WT or E342Q. 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112176-g006: ATPase activities of various streptavidin-purified samples.(A) Coomassie Blue staining after SDS-PAGE of purified wild-type complex (DWT-C), D560N variant (DD560N-C), E342Q variant (DE342Q-C), and wild-type Drs2p expressed alone. (B–C and E–F) The ATPase activity of the same samples (after 5-fold dilution resulting in about 60 µg/mL Drs2p in the case of the WT enzyme) was measured at 30°C in a KNG medium supplemented with 1 mg/mL DDM, 0.025 mg/mL PS and 1 mM Mg-ATP, in the absence (circles and dashed lines) or presence (triangles and continuous lines) of 0.025 mg/mL PI4P. The dotted line in panels C-F is given for easier comparison with results for WT. (D) Functional complementation of the temperature-sensitive phenotype of Δdrs2 yeast cells. Yeast cells, either wild-type or Δdrs2, were transformed with plasmids bearing DRS2 tagged at its 5′ end, either WT or E342Q. 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.
Mentions: As most of the ATP hydrolyzing contaminants in yeast membranes have presumably been removed during purification, the ATPase activity of the Drs2p-Cdc50p complex could be examined. At 30°C, we were indeed able to detect ATPase activity, and also to reveal, for the WT enzyme, its strong dependence on PI4P (Figure 6B).

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