522
PHOSPHOLIPID TRANSFER PROCESSES
[61]
Phosphatidylserine Transfer Protein The molecular weight of the PtdSer-TP as determined by SDS-polyacrylamide gel electrophoresis is 35K. Antibodies raised against the yeast Ptdlns-TP do not cross-react with the PtdSer-TP. The protein is cold-labile (50% inactivation after freezing) and is completely inactivated after heating to 50° for 10 rain or to 60 ° for 2 min. Treatment with the proteinases trypsin, proteinase K, and pronase completely abolishes transfer activity. Ca 2÷ and Mn 2÷ at 0.5 mM concentrations inhibit protein-catalyzed PtdSer transfer from phospholipid vesicles to mitochondria and between phospholipid vesicles. Phosphatidylserine is the preferred substrate, but other lipids are also transferred, the relative transfer rates being as follows: PtdSer 100%, PtdEtn 30%, cardiolipin 14%, phosphatidic acid 10%, ergosterol 10%. Inhibition of transfer by negatively charged phospholipids is similar to that observed for the Ptdlns-TP.
[61] P h o s p h o l i p i d T r a n s f e r P r o t e i n s f r o m H i g h e r P l a n t s B y C H A N T A L VERGNOLLE, V I N C E N T A R O N D E L , A L A I N JOLLIOT, and JEAN-CLAUDE KADER
Introduction
Plant cells contain soluble proteins able to facilitate in vitro bidirectional movements of phospholipids between membranes. 1,2 These proteins, called phospholipid transfer proteins (PLTP), have been purified and characterized from plant tissues and also from animal tissues or yeast (see [58]-[60], this volume). PLTPs are assumed to participate in the intracellular distribution of phospholipids and could be involved in membrane biogenesis or in the function of membrane-bound enzymes using lipids as substrates.l-4 An additional interest of PLTP is their ability to replace the endogenous phospholipids of a membrane by phospholipids originating from other membranes. PLTP can thus modify the lipid compo1 j. C. Kader, in "Subcellular Biochemistry" (H. J. Hilderson, ed.), Vol. 16, p. 69. Plenum, New York, 1990. 2 V. Arondel and J. C. Kader, Experientia 46, 579 (1990). 3 K. W. A. Wirtz, in "Lipid-Protein Interactions" (P. C. Jost and O. H. Griffith, eds.), p. 151. Elsevier, Amsterdam, 1982. 4 j. C. Kader, D. Douady, and P. Mazliak, in "Phospholipids, a Comprehensive Treatise" (J. N. Hawthorne and G. B. Ansell, eds.), p. 279. Elsevier, Amsterdam, 1982.
METHODS IN ENZYMOLOGY,VOL. 209
Copyright © 1992by AcademicPress, Inc. All rightsof reproductionin any form reserved.
[61]
PHOSPHOLIPID TRANSFER PROTEINS FROM PLANTS
523
sition of a membrane. This allows the study of the phospholipid-protein interactions within membranes and of the effects of changes in lipid concentrations on the functional properties of membranes. PLTP can also been used as mild agents to determine the asymmetry of the lipid composition of the membrane leaflets. Finally, these proteins are useful "tools" for inserting exogenous phospholipids, for example, those containing fluorescent compounds, in order to determine the mobility of lipids within membranes. 1-4 PLTP from plants are particularly suitable for these purposes because (1) the starting material (generally seeds or leaves) is abundant; (2) PLTP are stable for weeks; (3) plant PLTP have a broad specificity for phospholipids, allowing their use for a wide range of these lipids. In this chapter the procedure for the isolation of PLTP from sunflower seedlings is presented. The same general fractionation procedure has been used for other plant organs: maize 5 and castor bean 6 seedlings and spinach leaves .7
Assay
Reagents Buffer A: 0.4 M sucrose, 6 mM EDTA, 1 mM cysteine chloride, 8 mM 2-mercaptoethanol, 0.1 M Tris-HCl (pH 7.5) Buffer B: 0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl (pH 7.2) Egg yolk phosphatidylcholine Labeled compounds from Amersham (UK): myo-[2-3H]inositol (144 GBq/mmol); 1-[1-3H]ethanol-2-amine hydrochloride (370 GBq/ mmol); [methyl-3H]choline chloride (2.85 TBq/mmol); cholesteryl [1J4C]oleate (1.85 GBq/mmol) Principle. Liposomes, containing [3H]phosphatidylcholine (the phospholipid to be transferred) and cholesteryl [14C]oleate (not transferred by the protein), are incubated with mitochondria in the presence of various amounts of PLTP. The increase in 3H label in the mitochondria recovered by centrifugation indicates a transfer of phosphatidylcholine from liposomes to mitochondria. The 14Cradioactivity (generally low) indicates the extent of cross-contamination of mitochondria by intact liposomes. Other lipid transfer assays have been used either with other membrane systems 5 D. Douady, M. Grosbois, F. Guerbette, and J. C. Kader, Biochim. Biophys. Acta 710, 143 (1982). 6 S. Watanabe and M. Yamada, Biochim. Biophys. Acta 876, 116 (1986). 7 j. C. Kader, M. Julienne, and C. Vergnolle, Eur. J. Biochem. 139, 411 (1984).
524
PHOSPHOLIPID TRANSFER PROCESSES
[61]
(two categories of liposomes, 8 liposomes-chloroplast envelope 9) or with other phospholipids, such as spin-labeled (Devaux, unpublished) or fluorescent (Moreau, unpublished) ones. Isolation ofMitochondria. Mitochondria are isolated by sucrose gradient centrifugation from 4-day-old maize seedlings as previously described.5 The seedlings (generally 500 g of fresh weight) are ground in buffer A and the mitochondria are recovered and purified by different centrifugation steps, including sucrose gradient centrifugation. Mitochondrial pellets are suspended in buffer B at a final concentration of 12 mg protein/ml. Mitochondria can be kept up to 1 month at - 2 0 ° before use. Preparation of Labeled Phospholipids. [3H]Phosphatidylcholine, [3H]phosphatidylinositol, and [3H]phosphatidylethanolamine are prepared by incubating 20 g of potato tuber slices (1 mm thick) for 16 hr at 25 °, with constant shaking, either with [methyl-3H]choline chloride, [3H]ethanolamine, or myo-[2-3H]inositol. Generally, 3.7 MBq of each labeled compound is used. Labeled lipids are extracted as previously described. ~° Preparation ofLiposomes. Liposomes are prepared by mixing, for one assay, 260 nmol of egg yolk phosphatidylcholine, 1 nmol of cholesteryl [1-14C]oleate, and 10 nmol of labeled phospholipid (-740 Bq). Generally, the compounds necessary for 10 assays are introduced into a conical flask. After evaporating the solvent under a stream of nitrogen, 2.5 ml of buffer B is added. The flask is vigorously shaken, and the lipid dispersion is then introduced into a plastic tube and irradiated for 30 min by a Branson sonifier under a stream of nitrogen. The final volume is adjusted to 2.5 ml with buffer B. Transfer Assay. Phospholipid transfer activity is determined using 3H-/14C-labeled liposomes as donor membranes and mitochondria as acceptor ones. According to Fig. 1, the labeled liposomes are incubated with maize mitochondria in the absence or presence of PLTP. Incubation is started with the addition of the liposomes and is carried out with constant stirring. After centrifugation at 4°, the mitochondrial pellets are suspended in 0.5 ml of 1% (w/v) Triton X-100, and the suspension is counted with a Kontron (Zurich, Switzerland) scintillation system using 10 ml of toluene scintillation {0.4% (w/v) 2,5-diphenyloxazole, 0.01% (w/v) 1,4-bis[2-(5phenyloxazolyl)benzene]} containing 33% (v/v) Triton X-100. Units of Transfer. The transfer activity is expressed as the percentage of liposomal [3H]phosphatidylcholine recovered in mitochondria after ins F. Guerbette, D. Douady, M. Grosbois, and J. C. Kader, Physiol. Veg. 19, 467 (1981). 9 M. Miquel, M. A. Block, J. Joyard, A. J. Dome, J. P. Dubacq, J. C. Kader, and R. Douce, Biochim. Biophys. Acta 937, 219 (1987). l0 E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959).
[61]
PHOSPHOLIPID TRANSFER PROTEINS FROM PLANTS
oleSt(oe~.~5[14 ~] oleate
~
525
~l'tochoa~oatein )
Incubation in a total volume of 3 ml adjusted with buffer B (30"/30 rain)
Add ten ml of cold buffer B Centrifugation 10,000 g for 10 rain
Suspend mitochondrial pellets in 0.5 ml of I% Triton X-100 Determination of 3 H/14 C radioactivities FIG. I. Assay of phospholipid transfer protein (PLTP).
cubation in the presence of PLTP. This value is corrected for crosscontamination of mitochondria by liposomes by subtracting the percentage obtained in the absence of PLTP. The transfer activity is also calculated, from this corrected percentage of transfer, as nanomoles of phospholipids transferred by PLTP. One unit of PLTP is defined as the amount which transfers 1 nmol of phosphatidylcholine per minute, and the specific activity is expressed as units per milligram of protein. Purification Procedure
Reagents Buffer C: 1 mM phenylmethylsulfonyl fluoride in buffer A Buffer D: 8 mM 2-mercaptoethanol, 3 raM sodium azide, 10 ram potassium phosphate (pH 7.2)
526
PHOSPHOLIPID TRANSFER PROCESSES
[61]
TABLE I PU~FICATION OF PHOSPHOLIPID TRANSFER PROTEIN FROM SUNFLOWER SEEDLINGS
Step
Protein (mg)
Specific activity (units/mg protein)
Recovery (%)
Purification factor
Crude extract SephadexG-75 CM2 fraction SephadexG-50
6375 425 47 22
0.06 0.72 2.1 3.6
-80 25.6 20.7
-12 35 60
Buffer E: 0.25 M potassium phosphate in buffer D DEAE-Trisacryl (IBF, Paris, France) Sephadex G-50, Sephadex G-75, carboxymethyl-Sepharose CL-6B (Pharmacia, Uppsala, Sweden) Step 1: Preparation of Crude Extract. Sunflower seeds (Helianthus annuus var. Rodeo) (500 g) provided by CETIOM (Paris, France) are soaked for 3 hr and germinated at 30° on wet vermiculite for 4 days in darkness. The roots are discarded. The aerial parts are then ground in 1 liter of buffer C with a Waring blender for 15 sec at maximum speed. The pH is maintained at 7.5 by addition of 1 M Tris. The homogenate is then squeezed through two layers of Miracloth (Calbiochem, San Diego, CA) and centrifuged for 10,000 g for 20 min. The pH of the supernatant is then adjusted to 5.1 with 2 M HC1 in order to aggregate the residual membranes. The homogenate is then stirred for 15 min and centrifuged for 10,000 g for 20 rain. The pH of the supernatant is readjusted to 7.5. Proteins are allowed to precipitate by adding 52 g of (NH4)2SO 4 per 100 ml. Stirring is continued overnight, followed by centrifugation at 10,000 g for 20 min. The pellets are suspended in a minimal volume of buffer D, dialyzed against the same buffer for 5 hr, and centrifuged at 10,000 g for 20 min. The clear brownish supernatant is used as the crude extract. When assayed for lipid transfer, a specific activity of 0.06 units/mg of protein is found (Table I). Step 2: Gel Filtration through Sephadex G-75. The crude extract is loaded onto a column of Sephadex G-75 (70 × 5 cm) equilibrated with buffer D. The proteins are eluted with the same buffer at a rate of 80 ml/ hr. Fifteen-milliliter fractions are collected, and the activity is measured on 3 ml of each fraction. A major peak of activity is detected in the group of fractions 80 to 100 (Fig. 2A). The active fractions are pooled. The specific activity is 0.72 units/mg of protein with a purification factor of 12. Step 3: Ion-Exchange Chromatography. The active fractions from the Sephadex column are loaded onto a DEAE-Trisacryl column (25 x 2.6 cm) connected to a carboxymethyl-Sepharose column (25 x 2.6 cm) previously
[61]
527
PHOSPHOLIPID TRANSFER PROTEINS FROM PLANTS
1.5
A
0.75
7 o 1.0 O0 O4
PHOSPHOLIPID TRANSFER PROCESSES
[61]
Phosphatidylserine Transfer Protein The molecular weight of the PtdSer-TP as determined by SDS-polyacrylamide gel electrophoresis is 35K. Antibodies raised against the yeast Ptdlns-TP do not cross-react with the PtdSer-TP. The protein is cold-labile (50% inactivation after freezing) and is completely inactivated after heating to 50° for 10 rain or to 60 ° for 2 min. Treatment with the proteinases trypsin, proteinase K, and pronase completely abolishes transfer activity. Ca 2÷ and Mn 2÷ at 0.5 mM concentrations inhibit protein-catalyzed PtdSer transfer from phospholipid vesicles to mitochondria and between phospholipid vesicles. Phosphatidylserine is the preferred substrate, but other lipids are also transferred, the relative transfer rates being as follows: PtdSer 100%, PtdEtn 30%, cardiolipin 14%, phosphatidic acid 10%, ergosterol 10%. Inhibition of transfer by negatively charged phospholipids is similar to that observed for the Ptdlns-TP.
[61] P h o s p h o l i p i d T r a n s f e r P r o t e i n s f r o m H i g h e r P l a n t s B y C H A N T A L VERGNOLLE, V I N C E N T A R O N D E L , A L A I N JOLLIOT, and JEAN-CLAUDE KADER
Introduction
Plant cells contain soluble proteins able to facilitate in vitro bidirectional movements of phospholipids between membranes. 1,2 These proteins, called phospholipid transfer proteins (PLTP), have been purified and characterized from plant tissues and also from animal tissues or yeast (see [58]-[60], this volume). PLTPs are assumed to participate in the intracellular distribution of phospholipids and could be involved in membrane biogenesis or in the function of membrane-bound enzymes using lipids as substrates.l-4 An additional interest of PLTP is their ability to replace the endogenous phospholipids of a membrane by phospholipids originating from other membranes. PLTP can thus modify the lipid compo1 j. C. Kader, in "Subcellular Biochemistry" (H. J. Hilderson, ed.), Vol. 16, p. 69. Plenum, New York, 1990. 2 V. Arondel and J. C. Kader, Experientia 46, 579 (1990). 3 K. W. A. Wirtz, in "Lipid-Protein Interactions" (P. C. Jost and O. H. Griffith, eds.), p. 151. Elsevier, Amsterdam, 1982. 4 j. C. Kader, D. Douady, and P. Mazliak, in "Phospholipids, a Comprehensive Treatise" (J. N. Hawthorne and G. B. Ansell, eds.), p. 279. Elsevier, Amsterdam, 1982.
METHODS IN ENZYMOLOGY,VOL. 209
Copyright © 1992by AcademicPress, Inc. All rightsof reproductionin any form reserved.
[61]
PHOSPHOLIPID TRANSFER PROTEINS FROM PLANTS
523
sition of a membrane. This allows the study of the phospholipid-protein interactions within membranes and of the effects of changes in lipid concentrations on the functional properties of membranes. PLTP can also been used as mild agents to determine the asymmetry of the lipid composition of the membrane leaflets. Finally, these proteins are useful "tools" for inserting exogenous phospholipids, for example, those containing fluorescent compounds, in order to determine the mobility of lipids within membranes. 1-4 PLTP from plants are particularly suitable for these purposes because (1) the starting material (generally seeds or leaves) is abundant; (2) PLTP are stable for weeks; (3) plant PLTP have a broad specificity for phospholipids, allowing their use for a wide range of these lipids. In this chapter the procedure for the isolation of PLTP from sunflower seedlings is presented. The same general fractionation procedure has been used for other plant organs: maize 5 and castor bean 6 seedlings and spinach leaves .7
Assay
Reagents Buffer A: 0.4 M sucrose, 6 mM EDTA, 1 mM cysteine chloride, 8 mM 2-mercaptoethanol, 0.1 M Tris-HCl (pH 7.5) Buffer B: 0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl (pH 7.2) Egg yolk phosphatidylcholine Labeled compounds from Amersham (UK): myo-[2-3H]inositol (144 GBq/mmol); 1-[1-3H]ethanol-2-amine hydrochloride (370 GBq/ mmol); [methyl-3H]choline chloride (2.85 TBq/mmol); cholesteryl [1J4C]oleate (1.85 GBq/mmol) Principle. Liposomes, containing [3H]phosphatidylcholine (the phospholipid to be transferred) and cholesteryl [14C]oleate (not transferred by the protein), are incubated with mitochondria in the presence of various amounts of PLTP. The increase in 3H label in the mitochondria recovered by centrifugation indicates a transfer of phosphatidylcholine from liposomes to mitochondria. The 14Cradioactivity (generally low) indicates the extent of cross-contamination of mitochondria by intact liposomes. Other lipid transfer assays have been used either with other membrane systems 5 D. Douady, M. Grosbois, F. Guerbette, and J. C. Kader, Biochim. Biophys. Acta 710, 143 (1982). 6 S. Watanabe and M. Yamada, Biochim. Biophys. Acta 876, 116 (1986). 7 j. C. Kader, M. Julienne, and C. Vergnolle, Eur. J. Biochem. 139, 411 (1984).
524
PHOSPHOLIPID TRANSFER PROCESSES
[61]
(two categories of liposomes, 8 liposomes-chloroplast envelope 9) or with other phospholipids, such as spin-labeled (Devaux, unpublished) or fluorescent (Moreau, unpublished) ones. Isolation ofMitochondria. Mitochondria are isolated by sucrose gradient centrifugation from 4-day-old maize seedlings as previously described.5 The seedlings (generally 500 g of fresh weight) are ground in buffer A and the mitochondria are recovered and purified by different centrifugation steps, including sucrose gradient centrifugation. Mitochondrial pellets are suspended in buffer B at a final concentration of 12 mg protein/ml. Mitochondria can be kept up to 1 month at - 2 0 ° before use. Preparation of Labeled Phospholipids. [3H]Phosphatidylcholine, [3H]phosphatidylinositol, and [3H]phosphatidylethanolamine are prepared by incubating 20 g of potato tuber slices (1 mm thick) for 16 hr at 25 °, with constant shaking, either with [methyl-3H]choline chloride, [3H]ethanolamine, or myo-[2-3H]inositol. Generally, 3.7 MBq of each labeled compound is used. Labeled lipids are extracted as previously described. ~° Preparation ofLiposomes. Liposomes are prepared by mixing, for one assay, 260 nmol of egg yolk phosphatidylcholine, 1 nmol of cholesteryl [1-14C]oleate, and 10 nmol of labeled phospholipid (-740 Bq). Generally, the compounds necessary for 10 assays are introduced into a conical flask. After evaporating the solvent under a stream of nitrogen, 2.5 ml of buffer B is added. The flask is vigorously shaken, and the lipid dispersion is then introduced into a plastic tube and irradiated for 30 min by a Branson sonifier under a stream of nitrogen. The final volume is adjusted to 2.5 ml with buffer B. Transfer Assay. Phospholipid transfer activity is determined using 3H-/14C-labeled liposomes as donor membranes and mitochondria as acceptor ones. According to Fig. 1, the labeled liposomes are incubated with maize mitochondria in the absence or presence of PLTP. Incubation is started with the addition of the liposomes and is carried out with constant stirring. After centrifugation at 4°, the mitochondrial pellets are suspended in 0.5 ml of 1% (w/v) Triton X-100, and the suspension is counted with a Kontron (Zurich, Switzerland) scintillation system using 10 ml of toluene scintillation {0.4% (w/v) 2,5-diphenyloxazole, 0.01% (w/v) 1,4-bis[2-(5phenyloxazolyl)benzene]} containing 33% (v/v) Triton X-100. Units of Transfer. The transfer activity is expressed as the percentage of liposomal [3H]phosphatidylcholine recovered in mitochondria after ins F. Guerbette, D. Douady, M. Grosbois, and J. C. Kader, Physiol. Veg. 19, 467 (1981). 9 M. Miquel, M. A. Block, J. Joyard, A. J. Dome, J. P. Dubacq, J. C. Kader, and R. Douce, Biochim. Biophys. Acta 937, 219 (1987). l0 E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959).
[61]
PHOSPHOLIPID TRANSFER PROTEINS FROM PLANTS
oleSt(oe~.~5[14 ~] oleate
~
525
~l'tochoa~oatein )
Incubation in a total volume of 3 ml adjusted with buffer B (30"/30 rain)
Add ten ml of cold buffer B Centrifugation 10,000 g for 10 rain
Suspend mitochondrial pellets in 0.5 ml of I% Triton X-100 Determination of 3 H/14 C radioactivities FIG. I. Assay of phospholipid transfer protein (PLTP).
cubation in the presence of PLTP. This value is corrected for crosscontamination of mitochondria by liposomes by subtracting the percentage obtained in the absence of PLTP. The transfer activity is also calculated, from this corrected percentage of transfer, as nanomoles of phospholipids transferred by PLTP. One unit of PLTP is defined as the amount which transfers 1 nmol of phosphatidylcholine per minute, and the specific activity is expressed as units per milligram of protein. Purification Procedure
Reagents Buffer C: 1 mM phenylmethylsulfonyl fluoride in buffer A Buffer D: 8 mM 2-mercaptoethanol, 3 raM sodium azide, 10 ram potassium phosphate (pH 7.2)
526
PHOSPHOLIPID TRANSFER PROCESSES
[61]
TABLE I PU~FICATION OF PHOSPHOLIPID TRANSFER PROTEIN FROM SUNFLOWER SEEDLINGS
Step
Protein (mg)
Specific activity (units/mg protein)
Recovery (%)
Purification factor
Crude extract SephadexG-75 CM2 fraction SephadexG-50
6375 425 47 22
0.06 0.72 2.1 3.6
-80 25.6 20.7
-12 35 60
Buffer E: 0.25 M potassium phosphate in buffer D DEAE-Trisacryl (IBF, Paris, France) Sephadex G-50, Sephadex G-75, carboxymethyl-Sepharose CL-6B (Pharmacia, Uppsala, Sweden) Step 1: Preparation of Crude Extract. Sunflower seeds (Helianthus annuus var. Rodeo) (500 g) provided by CETIOM (Paris, France) are soaked for 3 hr and germinated at 30° on wet vermiculite for 4 days in darkness. The roots are discarded. The aerial parts are then ground in 1 liter of buffer C with a Waring blender for 15 sec at maximum speed. The pH is maintained at 7.5 by addition of 1 M Tris. The homogenate is then squeezed through two layers of Miracloth (Calbiochem, San Diego, CA) and centrifuged for 10,000 g for 20 min. The pH of the supernatant is then adjusted to 5.1 with 2 M HC1 in order to aggregate the residual membranes. The homogenate is then stirred for 15 min and centrifuged for 10,000 g for 20 rain. The pH of the supernatant is readjusted to 7.5. Proteins are allowed to precipitate by adding 52 g of (NH4)2SO 4 per 100 ml. Stirring is continued overnight, followed by centrifugation at 10,000 g for 20 min. The pellets are suspended in a minimal volume of buffer D, dialyzed against the same buffer for 5 hr, and centrifuged at 10,000 g for 20 min. The clear brownish supernatant is used as the crude extract. When assayed for lipid transfer, a specific activity of 0.06 units/mg of protein is found (Table I). Step 2: Gel Filtration through Sephadex G-75. The crude extract is loaded onto a column of Sephadex G-75 (70 × 5 cm) equilibrated with buffer D. The proteins are eluted with the same buffer at a rate of 80 ml/ hr. Fifteen-milliliter fractions are collected, and the activity is measured on 3 ml of each fraction. A major peak of activity is detected in the group of fractions 80 to 100 (Fig. 2A). The active fractions are pooled. The specific activity is 0.72 units/mg of protein with a purification factor of 12. Step 3: Ion-Exchange Chromatography. The active fractions from the Sephadex column are loaded onto a DEAE-Trisacryl column (25 x 2.6 cm) connected to a carboxymethyl-Sepharose column (25 x 2.6 cm) previously
[61]
527
PHOSPHOLIPID TRANSFER PROTEINS FROM PLANTS
1.5
A
0.75
7 o 1.0 O0 O4
