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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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Size-exclusion chromatography and mass spectrometry analysis of the purified Drs2p-Cdc50p complex.(A) Top: calibration of the TSK-3000SW silica gel column with gel filtration standards. The elution volume of Thyroglobulin corresponds to the dead volume (V0), while Vitamin B12 is eluted at a volume close to the total volume (Vt). Bottom: size-exclusion chromatography profile of the streptavidin-purified Drs2p-Cdc50p complex (continuous line). The streptavidin-purified and Ni2+-TED-treated fraction was first concentrated on YM100 filters and 300 µL was then injected on the column. Fractions 1–4 were collected. The dotted line shows the behavior of a control DDM-solubilized SR SERCA1a sample (500 µL at 4 mg/mL was injected). (B) Analysis of the collected fractions on an 8% Coomassie Blue stained SDS-PAGE. (C) An aliquot of the SEC-eluted sample was submitted to mass spectrometry analysis. Spectra were acquired on a range of m/z values from 30,000 to 200,000.
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pone-0112176-g004: Size-exclusion chromatography and mass spectrometry analysis of the purified Drs2p-Cdc50p complex.(A) Top: calibration of the TSK-3000SW silica gel column with gel filtration standards. The elution volume of Thyroglobulin corresponds to the dead volume (V0), while Vitamin B12 is eluted at a volume close to the total volume (Vt). Bottom: size-exclusion chromatography profile of the streptavidin-purified Drs2p-Cdc50p complex (continuous line). The streptavidin-purified and Ni2+-TED-treated fraction was first concentrated on YM100 filters and 300 µL was then injected on the column. Fractions 1–4 were collected. The dotted line shows the behavior of a control DDM-solubilized SR SERCA1a sample (500 µL at 4 mg/mL was injected). (B) Analysis of the collected fractions on an 8% Coomassie Blue stained SDS-PAGE. (C) An aliquot of the SEC-eluted sample was submitted to mass spectrometry analysis. Spectra were acquired on a range of m/z values from 30,000 to 200,000.

Mentions: After concentration on a 100 kDa centrifugal filter device, the purified Drs2p-Cdc50p sample was analyzed by SEC on a TSK-3000SW silica gel column, in the presence of DDM. Judging from the elution positions of soluble standards (Figure 4A, top) or of DDM-solubilized SERCA1a (110 kDa, 5.5 nm Stokes radius, Figure 4A, bottom), the main elution peak (Figure 4A, bottom) was found in the expected region. Its fairly symmetrical shape indicated a good homogeneity and the initial sample apparently contained only a very limited amount of aggregated material eluting in the column void volume. Cdc50p eluted together with Drs2p, as judged from SDS-PAGE analysis of eluted fractions, confirming the tight association of the two protein subunits (Figure 4B).


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)

Size-exclusion chromatography and mass spectrometry analysis of the purified Drs2p-Cdc50p complex.(A) Top: calibration of the TSK-3000SW silica gel column with gel filtration standards. The elution volume of Thyroglobulin corresponds to the dead volume (V0), while Vitamin B12 is eluted at a volume close to the total volume (Vt). Bottom: size-exclusion chromatography profile of the streptavidin-purified Drs2p-Cdc50p complex (continuous line). The streptavidin-purified and Ni2+-TED-treated fraction was first concentrated on YM100 filters and 300 µL was then injected on the column. Fractions 1–4 were collected. The dotted line shows the behavior of a control DDM-solubilized SR SERCA1a sample (500 µL at 4 mg/mL was injected). (B) Analysis of the collected fractions on an 8% Coomassie Blue stained SDS-PAGE. (C) An aliquot of the SEC-eluted sample was submitted to mass spectrometry analysis. Spectra were acquired on a range of m/z values from 30,000 to 200,000.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112176-g004: Size-exclusion chromatography and mass spectrometry analysis of the purified Drs2p-Cdc50p complex.(A) Top: calibration of the TSK-3000SW silica gel column with gel filtration standards. The elution volume of Thyroglobulin corresponds to the dead volume (V0), while Vitamin B12 is eluted at a volume close to the total volume (Vt). Bottom: size-exclusion chromatography profile of the streptavidin-purified Drs2p-Cdc50p complex (continuous line). The streptavidin-purified and Ni2+-TED-treated fraction was first concentrated on YM100 filters and 300 µL was then injected on the column. Fractions 1–4 were collected. The dotted line shows the behavior of a control DDM-solubilized SR SERCA1a sample (500 µL at 4 mg/mL was injected). (B) Analysis of the collected fractions on an 8% Coomassie Blue stained SDS-PAGE. (C) An aliquot of the SEC-eluted sample was submitted to mass spectrometry analysis. Spectra were acquired on a range of m/z values from 30,000 to 200,000.
Mentions: After concentration on a 100 kDa centrifugal filter device, the purified Drs2p-Cdc50p sample was analyzed by SEC on a TSK-3000SW silica gel column, in the presence of DDM. Judging from the elution positions of soluble standards (Figure 4A, top) or of DDM-solubilized SERCA1a (110 kDa, 5.5 nm Stokes radius, Figure 4A, bottom), the main elution peak (Figure 4A, bottom) was found in the expected region. Its fairly symmetrical shape indicated a good homogeneity and the initial sample apparently contained only a very limited amount of aggregated material eluting in the column void volume. Cdc50p eluted together with Drs2p, as judged from SDS-PAGE analysis of eluted fractions, confirming the tight association of the two protein subunits (Figure 4B).

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