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Lipid requirements of the plasma membrane ATPases from oat roots and yeast
Plant Science, 56 (1988) 117-122 Elsevier Scientific Publishers Ireland Ltd.
117
LIPID REQUIREMENTS OF THE P L A S M A M E M B R A N E ATPases FROM OAT ROOTS AND YEAST RAMON SERRANO, CONSUELO MONTESINOS and JUAN SANCHEZ*
lnstituto de Investigaciones Biomedicas del CSIC, Arzobispo Mwrcillo 4, ~80~9 Madrid ?SpainJand European Molecular Biology Laboratory, Meyerhofstrasse 1, 6900 Heidelberg ~F.R.G.) (Received November 18th, 1987) (Revision received March 2nd, 1988) (Accepted March 2nd, 1988) The lipid specificity of the plasma membrane ATPases from oat roots and yeast has been investigated by reconstituting delipidatod enzyme with phospholipid vesicles and with micelles of lysophospholipids and other detergents. The plant ATPase is activated by Triton X-100 and by all phospholipid and lysophospholipid species, exhibiting only a slight preference for zwittorionlc polar heads (phoaphorylcholine and phosphorylethanolamine). No unsaturation is required on the hydrophobic chain. On the other hand, the yeast ATPase requires a negatively charged polar head (with preference for phosphorylglycerol and phosphorylinositol) and an unsaturated bydrophobic chain.
Key words: plasma membrane; ATPase; lipid
Introduction The activity of many membrane enzymes is dependent on lipids, although the degree of specificity for this requirement is quite variable [1]. This lipid specificity may be important for the physiological regulation of membrane enzymes, as indicated by the activation of p r o tein kinase C by diacylglycerol [2]. In addition, the plausibility of expressing the gene of a membrane enzyme in a different organism will depend on the lipid compatibility of the host membrane. Both aspects are relevant for the protonpumping ATPases of fungal and plant plasma *Present address: Institut fur Biochemie der Pflanzen, Untore Karspule 2, 3400 Gottingen, F.R.G. Abbreviations: CH, cholic acid; LPA, lysophosphatidic acidoleoyl LPC, lysophosphatidylcholine; LPC', lysophosphatidylcholine-oleoyl; LPE, lysophosphatidylethanolamine; LPG, lysophosphatidylglycerol; LPI, lysophosphatidylinositol; LPS, lysophosphatidylserine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, pbesphatidylethanolamine; PG, phosphatidylglycerol; PI, phoaphatidylinositoh PS, pbosphatidylserine; SB, crude soybean phospholipids; TX, Triton X-100.
membranes [3]. These enzymes are activated by growth-promoting factors such as glucose in yeast [4] and auxin and fusicoccin in plants [5]. The mechanism of activation is unknown and the generation of an activating lipid is a possible one. On the other hand the gene for the yeast plasma membrane ATPase has been isc~ lated [6] and the gene for the oat root enzyme would also be available in the near future (J.M. Pardo and R. Serrano, in prep.). The expression of these genes in other organisms may be a powerful tool to clarify the physiological role of proton pumping and is currently being pursued in our laboratory. Therefore, it was important to clarify the lipid requirements of the yeast and oat root ATPases and to explore the formation of activating lipids during the physiological regulation of the enzymes. Methods
Enzyme preparations The plasma membrane ATPase from the yeast Saccharomyces cerevisiae was purified as described [7], except that Zwittergent-14 was not added to the glycerol gradient. The
0168-9452/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
118
plasma membrane ATPase from oat (Avena sativa) roots was purified as described [8] but omitting the solubilization and precipitation steps. The two enzyme preparations were delipidated by treatment with cholate. The incubation contained 1 mg/ml protein, 20 mg/ml cholate, 0.4 M ammonium sulfate, 16% glycerol, 8 mM Tris (pH 7.7 with HC1), 0.8 mM EDTA and 0.8 mM dithioerythritol. After 10 min at room temperature, 1 ml of the incubation mixture was layered over a cushion of 5 ml with 36% glycerol and Tris, EDTA and dithioerythritol as above and centrifuged for 3 h at 40000 rev./ rain in a Beckman 65 rotor. The pellet was homogenized with 6 ml of 200/0 glycerol containing Tris, EDTA and dithioerythritol and centrifuged for 1.5 h as above. Yeast plasma membranes for lipid extraction were prepared as described [9]. Membranes from glucose-activated yeast cells were obtained by treating the cells with 20/o glucose for 5 rain at room temperature before homogenization [4].
Lipid preparations The following purified phospholipids were obtained from Sigma: Phosphatidylcholine {from egg yolk) (PC), phosphatidylethanolamine (from soybean) (PE), phosphatidylinositol (from soybean) (PI), phosphatidylserine (from bovine brain) (PS), phosphatidylglycerol (from egg yolk) (PG), phosphatidic acid (from egg yolk) (PA), lysophosphatidylcholine (from egg yolk, mostly palmitoyl and stearoyl) (LPC), lysophosphatidylcholine~leoyl (LPC'C), lysophosphatidylethanolamine {from egg yolk, mostly stearoyl and palmitoyl) (LPE), lysophosphatidylinositol (from soybean, mostly palmitoyl and stearol) (LPI), lysophosphatidylserine {from bovine brain, mostly palmitoyl and stearoyl) (LPS), lysophosphatidylglycerol (from egg yolk, mostly palmitoyl and stearoyl) (LPG), lysophosphatidic acid-oleoyl (LPA). Triton X-100 (TX) was obtained from Merck, cholic acid (CH) from Sigma was purifed as described [10] and neutralized with NaOH,
crude soybean phospholipids ( S B ) were obtained from Sigma as phosphatidylcholine type II-S and purified as described [10]. Crude lipid preparations from yeast plasma membranes were obtained by a modified Bligh and Dyer procedure [11] after precipitation of the samples with trichloroacetic acid to improve the extraction of acidic phospholipids. In order to increase recovery of lysophospholipids, extraction with butanol was also employed [12]. Lipid composition of membrane extracts was analyzed by thin-layer chromatography as described [13,14]. Lipid solutions were prepared in 10 mM Tris (pH 8 with HCI) and 1 mM EDTA. Phospholipids were sonicated to clarity in a Branson sonicator. Lysophospholipids were heated at 56 °C to dissolve.
Assay of A TPase activity The enzyme preparations ( 5 - 1 0 ~g protein) were incubated in 1 ml of buffer containing 50 mM 4-morpholine ethanesulfonic acid, 5 mM MgSO 4, 50 mM K N O 3, 5 mM sodium azide and 0.2 mM ammonium molybdate. The pH was adjusted with Tris to either 5.7 (yeast ATPase) or 6.5 (oat root ATPase). Phospholipids (100 ~g), lysophospholipids (10 ~g), cholate and TX (50 ~g) were preincubated with the enzymes for 5 min at 30 °C before starting the reaction with ATP. Pi production was determined as described [9l. Reconstitution of proteoliposomes Direct incorporation of the enzymes into preformed liposomes was effected by mixing in assay buffer as described above. The freezethaw sonication method, in the presence of 150 mM KC1, was also employed [8]. Pro tein de termination The Bio-Rad Protein Assay Reagent was employed, with bovine globulin as standard. Results
Me thodological considerations We have investigated the lipid specificity of plasma membrane ATPases by delipidating the
119
enzyme preparations with cholate, washing the cholate exhaustively and reconstituting the enzymes with different lipids. Reconstitution with phospholipid vesicles was effected by two different procedures because with some membrane enzyme preparations the lipid specificity reflects the needs of the reeonstitution procedure [15]. Freeze-thaw sonication and direct incorporation of the ATPases by mixing with preformed liposomes gave essentially similar results, suggesting that the observed lipid specificity corresponds to the requirements of the enzymes. Only the results obtained by the later procedure are presented below. In addition, we have reconstituted the ATPases with lysophospholipids and other detergents by simple mixing. The lysophospholipids have similar structures to membrane phospholipids and do not have the possible complications of the reconstitution procedure because they do not form bilayers. The concentration of lipids indicated in Methods was in the optimal range of titration experiments.
Lipid specificity of the oat root A TPase Optimal stimulation of the oat root plasma membrane ATPase was obtained by LPC (Fig. 1). LPE was slightly less effective and the lysophospholipids with acidic polar heads (LPI, LPS, LPG and LPA) and TX produced less activation. Cholate was ineffective. A similar preference for zwitterionic polar heads was observed in the case of phospholipids, with PC and PE producing more activation than the acidic phospholipids PI, PS, PG and PA. On the other hand, saturated (LPC) and unsaturated (LPC') lysophospholipids were equally effective. These results indicate that the oat root ATPase is only moderately specific in terms of its lipid requirements. The higher efficiency of LPC over PC may be due to a more complete interaction of the ATPase with the detergent than with the phospholipid vesicles. Accordingly, the activity with PC can be slightly increased (about 30%) by inclusion of cholate (data not shown).
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Lipid Fig. 1. Activation of the oat root plasma membrane A T P a s e by different lipids. For the lipid abbreviations see Methods. The activity with soybean lipids (SB), taken as 100o/o, w a s 1.4 tanol • rain -~ • m g protein -~. Results of a typical experiment are shown and correspond to the average of duplicates differing in less than 5%.
120
Lipid specificity of the yeas t A TPase Only acidic phospholipids are effective in activating the yeast plasma membrane ATPase (Fig. 2). PG and PI produce the highest activity, with PS and PA being less effective. A requirement for unsaturated hydrophobic chains is suggested by the lack of activation by saturated LPG and LPI while oleoyl LPA was effective. Also, Triton X-100 and cholate were completely inactive. Therefore the yeast ATPase seems to be much more lipid specific than the oat root ATPase. Lack of changes in plasma membrane lipids during activation of the yeast A TPase Yeast cells were treated with glucose for 5 rain, homogenized and lipids extracted from purified plasma membranes exhibiting an activated state of the ATPase [4]. Control membranes were prepared from cells not incubated with glucose. No significant difference in the major plasma membrane phospholipids (PI, PC and PE, Ref. 16) was detected by thin-layer chroma-
tography (data not shown). In addition, lipid extracts from both plasma membrane preparations activated the ATPase to a similar extent (Table I). Therefore, we do not find evidence for the direct participation of lipids in the activation of the yeast plasma membrane ATPase triggered by glucose. Discussion
The lipid specificies of the oat root and yeast plasma membrane ATPase are completely different. The plant enzyme is relatively unspecific. Even Triton X-100 activates partially, although optimal activity is obtained with lipids containing a zwitterionic polar head. On the other hand, the yeast enzyme requires both a negatively charged polar head (with preference for phosphorylinositol and phosphorylglycerol) and an unsaturated hydrophobic chain. However, even the requirements of the yeast enzyme should be easily satisfied by any biological membrane. Accordingly, good activation
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Fig. 2. Activation of t h e y e a s t plasma m e m b r a n e A T P a s e by different lipids. The activity with soybean lipids (SB), taken as 100%, was 2.6 Aanol • min -1- m g p r o t e i n -1. O t h e r details as in Fig. 1.
121 Table I. Activation of the yeast plasma membrane ATPase by lipids extracted from yeast plasma membranes. 100 ~g lipids w e r e mixed with 10 ~g enzyme as described in Methods. Lipid source
-
Soybean Plasmamembranesfrom glucose-fermentingyeast Plasmamembranesfrom glucose-starvedyeast
Extraction procedure
ATPase activity (umol/min"mg protein-z)
Bligband Dyer Butanol Blighand Dyer Butanol
0.06 1.4 0.8 1.0 0.7 1.1
has been observed with crude phospholipid preparations from such diverse systems as soybean (Fig. 2), yeast plasma membranes, bovine brain and Escherichia coli (data not shown). Therefore, during the expression of the yeast plasma membrane ATPase in other organisms no problems are expected with the lipid composition of the new host membranes. The utilization of lysophospholipids and, in the case of phospholipids, of different reconstitution procedures greatly facilitates the interpretation of lipid specificity studies. In previous work with plant plasma membrane ATPases these precautions were not adopted and contradictory results were obtained. An ATPase preparation from radish (Raphanus sati~ts) seedlings was activated by PI but neither PC nor PE were effective [17]. On the other hand, an enzyme from mung beans (Vigna radiata) was activated by PC, PS and PG but neither PI nor PE were effective [18]. LPC was effective in both preparations but as other lysophospholipids were not tested it is not clear to what extent the observed specificity reflects the reconstitution requirements of these enzyme preparations. A detailed study of the lipid specificity of the plasma membrane ATPase from Schizosaccharomyces pombe [19] indicated that all phospholipid species activated to a similar extent and that saturated and unsaturated LPC were also equivalent. Therefore this enzyme has the same poor lipid specificity as the plant
ATPase. On the other hand, the enzyme from another fungus, Neurospora crassa, seems to require negatively charged phospholipids [20], resembling the yeast (Saccharomyces) ATPase. From previously studied membrane enzymes, the (Na/K) ATPase from animal plasma membranes exhibits the same requirements for negatively charged polar heads and unsaturated hydrophobie chains as the yeast ATPase [21,22]. On the other hand, the (Ca) ATPase from sarcoplasmic reticulum can be activated by detergents such as Triton X-100 and Brij 52 [23] and by all phospholipid species, although with slight preference for PC [24]. Therefore this latter enzyme has a similar lipid specificity as the plant ATPase. These results and the experiments performed with lipids extracted from yeast plasma membranes make it unlikely that the activation of the yeast and plant ATPases by growthpromoting factors [ 3 - - 5 ] were mediated by the generation of an activating lipid. Recent reports on lipid changes induced by glucose in yeast [25] and by auxins in plants [26,27] may be related to the activation of other proteins connected with the growth response. Acknowledgements This work was partially financed by a grant of the Spanish Comision Asesora de Investigacion Cientifica y Teeniea. J.S. was a fellow of the Fundacion March.
122 References 1 2 3 4
5
6
7
8
9
10
11
12
13
14 15
H. Sanderman, Regulation of membrane enzymes by lipids. Biochim. Biophys. Acta, 515 (1978) 209--237. Y Nishizuka, Studies and perspectives of protein kinase C. Science, 233 (1986) 305-312. R. Serrano, Plasma Membrane ATPase of Plants and Fungi, CRC Press, Boca Raton, Florida, 1985. R. Serrano, In vivo glucose activation of the yeast plasma membrane ATPase. FEBS Lett., 158 (1933) 11--14. E. Marre, Integration of solute transport in cereals, in: D.L. Laidman and R.G. Wyn Jones (Eds.), Recent Advances in the Biochemistry of Cereals, Academic Press, 1979, pp. 3 - 25. R. Serrano, M.C. Kielland-Brandt and G.R. Fink, Yeast plasma membrane ATPase is essential for growth and has homology with (Na÷ + K*), K ÷ and Ca2*-ATPases. Nature, 319 (1986) 689--693. F. Malpartida and R. Serrano, Phosphorylated intermediate of the ATPase from the plasma membrane of yeast. Eur. J. Biochem., 116 (1981} 413--417. F. Vara and R. Serrano, Partial purification and properties of the proton-translocating ATPase of plant plasma membranes. J. Biol. Chem., 257 (1982) 12826-12830. R. Serrano, Characterization of the plasma membrane (ATPase of Saccharomyces cerevisiae. Mol. Cell. Biochem., 22 (1978) 51 --62. Y. Kagawa and E. Racker, Reconstitution of vesicles catalyzing ~Pi~ATP exchange. J. Biol. Chem., 246 (1971) 5477 -- 5487. K.D. Gibson, J.D. Wilson and S. Udenh'iend, The enzymatic conversion of pbospholipid ethanolamine to phospholipid choline in rat liver. J. Biol. Chem., 236 (1961) 673-- 679. K.S. Bjerve, L.N.W. Daae and J. Bremer, The selective loss of lysophospholipids in some commonly used lipidextraction procedures. Anal. Biochem., 58 (1974) 238245. J.C. Dittmer and M.A. Wells, Quantitative and qualitative analysis of lipids and lipid components. Methods Enzymol., 14 (1969) 482-- 530. V.P. Skipski and M. Barclay, Thin-layer chromatography of lipids. Methods Enzymol., 14 (1969) 530- 598. G.D. Eytan, Use of liposomes for reconstitution of biological functions. Biochim. Biophys. Acta, 694 (1982) 185-- 202.
16
17
18
19
20
21
22
23
24
25
26
27
S.A. Henry, Membrane lipids of yeast: biochemical and genetic studies, in: J.N. Strathern, E.W. Jones and J.R. Broach (Eds.), The Molecular Biology of the Yeast Saccharomyces. Metabolism and Gene Expression, Cold Spring Harbor Laboratory, 1982, pp. 101 - 158. M.C. Cocueci and E. Marre, Lysophosphatidylcholineactivated, vanadata-inhibited, Mg-ATPase from radish microsomes. Biochim. Biophys. Acta, 771 (1984) 4 2 - 52. K. Kasamo and I. Nouchi, The role of phospholipids in plasma membrane ATPase activity in Vigna radiata L. (mung bean) roots and hypocotyls. Plant Physiol., 83 (1987) 323-- 328. J.P. Dufour and A. Goffeau, Phospholipid reactivation of the purified plasma membrane ATPase of yeast. J. Biol. Chem., 255 (1980) 10591 - 10598. G.A. Scarborough, Properties of the Neurospora crassa plasma membrane ATPase. Arch. Biochem. Biophys., 180 (1977} 384-- 393. H.K. Kimelberg and D. Papahadjopoulos, Phospbolipid requirements for (Na+K) ATPase activity: head group specificity and fatty acid fluidity. Biochim. Biophys. Aeta, 282 (1972} 277-- 292. B. Roolofsen and L.L.M. Van Deenen, Lipid requirement of membrane-bound ATPase. Studies on human erythrocyte ghosts. Eur. J. Biochem., 40 (1973) 245257. V.I. Melgunov and E.I. Akimova, The dependence for reactivation of lipid-depleted Ca-ATPase of sarc~ plasmic reticulum by non-ionic detergents on their hydrophfle/lipophfle balance. FEBS Lett., 121 (1980) 235-- 238. J.P. Bennet, G.A. Smith, M.D. Houslay, T.R. Hesketh, J.C. Metcalfe and G.B. Warren, The pbospholipid head group specificity of an ATP-dependent calcium pump. Biochim. Biophys. Acta, 513 (1976) 310--320. K. Kaibuchi, A. Miyajima, K. Arai and K. Matsumoto, Possible involvement of RAS-encoded proteins in glucose-induced inositolphospbolipid turnover in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A., 83 (1986} 8172- 8176. D.J. Morre, B. Gripshover, A. Monroe and J.T. Morre, Phosphatidylinositol turnover in isolated soybean membranes stimulated by the synthetic growth hormone 2,4-dichlorophenoxyacetic acid. J. Biol. Chem., 259 (1984} 15364-- 15868. C. Ettlinger and L. Lehle, Auxin induces rapid changes in phosphatidylinositol metabolites. Nature, 331 (1988) 176-178.
117
LIPID REQUIREMENTS OF THE P L A S M A M E M B R A N E ATPases FROM OAT ROOTS AND YEAST RAMON SERRANO, CONSUELO MONTESINOS and JUAN SANCHEZ*
lnstituto de Investigaciones Biomedicas del CSIC, Arzobispo Mwrcillo 4, ~80~9 Madrid ?SpainJand European Molecular Biology Laboratory, Meyerhofstrasse 1, 6900 Heidelberg ~F.R.G.) (Received November 18th, 1987) (Revision received March 2nd, 1988) (Accepted March 2nd, 1988) The lipid specificity of the plasma membrane ATPases from oat roots and yeast has been investigated by reconstituting delipidatod enzyme with phospholipid vesicles and with micelles of lysophospholipids and other detergents. The plant ATPase is activated by Triton X-100 and by all phospholipid and lysophospholipid species, exhibiting only a slight preference for zwittorionlc polar heads (phoaphorylcholine and phosphorylethanolamine). No unsaturation is required on the hydrophobic chain. On the other hand, the yeast ATPase requires a negatively charged polar head (with preference for phosphorylglycerol and phosphorylinositol) and an unsaturated bydrophobic chain.
Key words: plasma membrane; ATPase; lipid
Introduction The activity of many membrane enzymes is dependent on lipids, although the degree of specificity for this requirement is quite variable [1]. This lipid specificity may be important for the physiological regulation of membrane enzymes, as indicated by the activation of p r o tein kinase C by diacylglycerol [2]. In addition, the plausibility of expressing the gene of a membrane enzyme in a different organism will depend on the lipid compatibility of the host membrane. Both aspects are relevant for the protonpumping ATPases of fungal and plant plasma *Present address: Institut fur Biochemie der Pflanzen, Untore Karspule 2, 3400 Gottingen, F.R.G. Abbreviations: CH, cholic acid; LPA, lysophosphatidic acidoleoyl LPC, lysophosphatidylcholine; LPC', lysophosphatidylcholine-oleoyl; LPE, lysophosphatidylethanolamine; LPG, lysophosphatidylglycerol; LPI, lysophosphatidylinositol; LPS, lysophosphatidylserine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, pbesphatidylethanolamine; PG, phosphatidylglycerol; PI, phoaphatidylinositoh PS, pbosphatidylserine; SB, crude soybean phospholipids; TX, Triton X-100.
membranes [3]. These enzymes are activated by growth-promoting factors such as glucose in yeast [4] and auxin and fusicoccin in plants [5]. The mechanism of activation is unknown and the generation of an activating lipid is a possible one. On the other hand the gene for the yeast plasma membrane ATPase has been isc~ lated [6] and the gene for the oat root enzyme would also be available in the near future (J.M. Pardo and R. Serrano, in prep.). The expression of these genes in other organisms may be a powerful tool to clarify the physiological role of proton pumping and is currently being pursued in our laboratory. Therefore, it was important to clarify the lipid requirements of the yeast and oat root ATPases and to explore the formation of activating lipids during the physiological regulation of the enzymes. Methods
Enzyme preparations The plasma membrane ATPase from the yeast Saccharomyces cerevisiae was purified as described [7], except that Zwittergent-14 was not added to the glycerol gradient. The
0168-9452/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
118
plasma membrane ATPase from oat (Avena sativa) roots was purified as described [8] but omitting the solubilization and precipitation steps. The two enzyme preparations were delipidated by treatment with cholate. The incubation contained 1 mg/ml protein, 20 mg/ml cholate, 0.4 M ammonium sulfate, 16% glycerol, 8 mM Tris (pH 7.7 with HC1), 0.8 mM EDTA and 0.8 mM dithioerythritol. After 10 min at room temperature, 1 ml of the incubation mixture was layered over a cushion of 5 ml with 36% glycerol and Tris, EDTA and dithioerythritol as above and centrifuged for 3 h at 40000 rev./ rain in a Beckman 65 rotor. The pellet was homogenized with 6 ml of 200/0 glycerol containing Tris, EDTA and dithioerythritol and centrifuged for 1.5 h as above. Yeast plasma membranes for lipid extraction were prepared as described [9]. Membranes from glucose-activated yeast cells were obtained by treating the cells with 20/o glucose for 5 rain at room temperature before homogenization [4].
Lipid preparations The following purified phospholipids were obtained from Sigma: Phosphatidylcholine {from egg yolk) (PC), phosphatidylethanolamine (from soybean) (PE), phosphatidylinositol (from soybean) (PI), phosphatidylserine (from bovine brain) (PS), phosphatidylglycerol (from egg yolk) (PG), phosphatidic acid (from egg yolk) (PA), lysophosphatidylcholine (from egg yolk, mostly palmitoyl and stearoyl) (LPC), lysophosphatidylcholine~leoyl (LPC'C), lysophosphatidylethanolamine {from egg yolk, mostly stearoyl and palmitoyl) (LPE), lysophosphatidylinositol (from soybean, mostly palmitoyl and stearol) (LPI), lysophosphatidylserine {from bovine brain, mostly palmitoyl and stearoyl) (LPS), lysophosphatidylglycerol (from egg yolk, mostly palmitoyl and stearoyl) (LPG), lysophosphatidic acid-oleoyl (LPA). Triton X-100 (TX) was obtained from Merck, cholic acid (CH) from Sigma was purifed as described [10] and neutralized with NaOH,
crude soybean phospholipids ( S B ) were obtained from Sigma as phosphatidylcholine type II-S and purified as described [10]. Crude lipid preparations from yeast plasma membranes were obtained by a modified Bligh and Dyer procedure [11] after precipitation of the samples with trichloroacetic acid to improve the extraction of acidic phospholipids. In order to increase recovery of lysophospholipids, extraction with butanol was also employed [12]. Lipid composition of membrane extracts was analyzed by thin-layer chromatography as described [13,14]. Lipid solutions were prepared in 10 mM Tris (pH 8 with HCI) and 1 mM EDTA. Phospholipids were sonicated to clarity in a Branson sonicator. Lysophospholipids were heated at 56 °C to dissolve.
Assay of A TPase activity The enzyme preparations ( 5 - 1 0 ~g protein) were incubated in 1 ml of buffer containing 50 mM 4-morpholine ethanesulfonic acid, 5 mM MgSO 4, 50 mM K N O 3, 5 mM sodium azide and 0.2 mM ammonium molybdate. The pH was adjusted with Tris to either 5.7 (yeast ATPase) or 6.5 (oat root ATPase). Phospholipids (100 ~g), lysophospholipids (10 ~g), cholate and TX (50 ~g) were preincubated with the enzymes for 5 min at 30 °C before starting the reaction with ATP. Pi production was determined as described [9l. Reconstitution of proteoliposomes Direct incorporation of the enzymes into preformed liposomes was effected by mixing in assay buffer as described above. The freezethaw sonication method, in the presence of 150 mM KC1, was also employed [8]. Pro tein de termination The Bio-Rad Protein Assay Reagent was employed, with bovine globulin as standard. Results
Me thodological considerations We have investigated the lipid specificity of plasma membrane ATPases by delipidating the
119
enzyme preparations with cholate, washing the cholate exhaustively and reconstituting the enzymes with different lipids. Reconstitution with phospholipid vesicles was effected by two different procedures because with some membrane enzyme preparations the lipid specificity reflects the needs of the reeonstitution procedure [15]. Freeze-thaw sonication and direct incorporation of the ATPases by mixing with preformed liposomes gave essentially similar results, suggesting that the observed lipid specificity corresponds to the requirements of the enzymes. Only the results obtained by the later procedure are presented below. In addition, we have reconstituted the ATPases with lysophospholipids and other detergents by simple mixing. The lysophospholipids have similar structures to membrane phospholipids and do not have the possible complications of the reconstitution procedure because they do not form bilayers. The concentration of lipids indicated in Methods was in the optimal range of titration experiments.
Lipid specificity of the oat root A TPase Optimal stimulation of the oat root plasma membrane ATPase was obtained by LPC (Fig. 1). LPE was slightly less effective and the lysophospholipids with acidic polar heads (LPI, LPS, LPG and LPA) and TX produced less activation. Cholate was ineffective. A similar preference for zwitterionic polar heads was observed in the case of phospholipids, with PC and PE producing more activation than the acidic phospholipids PI, PS, PG and PA. On the other hand, saturated (LPC) and unsaturated (LPC') lysophospholipids were equally effective. These results indicate that the oat root ATPase is only moderately specific in terms of its lipid requirements. The higher efficiency of LPC over PC may be due to a more complete interaction of the ATPase with the detergent than with the phospholipid vesicles. Accordingly, the activity with PC can be slightly increased (about 30%) by inclusion of cholate (data not shown).
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Lipid Fig. 1. Activation of the oat root plasma membrane A T P a s e by different lipids. For the lipid abbreviations see Methods. The activity with soybean lipids (SB), taken as 100o/o, w a s 1.4 tanol • rain -~ • m g protein -~. Results of a typical experiment are shown and correspond to the average of duplicates differing in less than 5%.
120
Lipid specificity of the yeas t A TPase Only acidic phospholipids are effective in activating the yeast plasma membrane ATPase (Fig. 2). PG and PI produce the highest activity, with PS and PA being less effective. A requirement for unsaturated hydrophobic chains is suggested by the lack of activation by saturated LPG and LPI while oleoyl LPA was effective. Also, Triton X-100 and cholate were completely inactive. Therefore the yeast ATPase seems to be much more lipid specific than the oat root ATPase. Lack of changes in plasma membrane lipids during activation of the yeast A TPase Yeast cells were treated with glucose for 5 rain, homogenized and lipids extracted from purified plasma membranes exhibiting an activated state of the ATPase [4]. Control membranes were prepared from cells not incubated with glucose. No significant difference in the major plasma membrane phospholipids (PI, PC and PE, Ref. 16) was detected by thin-layer chroma-
tography (data not shown). In addition, lipid extracts from both plasma membrane preparations activated the ATPase to a similar extent (Table I). Therefore, we do not find evidence for the direct participation of lipids in the activation of the yeast plasma membrane ATPase triggered by glucose. Discussion
The lipid specificies of the oat root and yeast plasma membrane ATPase are completely different. The plant enzyme is relatively unspecific. Even Triton X-100 activates partially, although optimal activity is obtained with lipids containing a zwitterionic polar head. On the other hand, the yeast enzyme requires both a negatively charged polar head (with preference for phosphorylinositol and phosphorylglycerol) and an unsaturated hydrophobic chain. However, even the requirements of the yeast enzyme should be easily satisfied by any biological membrane. Accordingly, good activation
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~/, v//A V//A
V//.4
None SB
PC
PE
PI
PS
PG
PA
LPC LPC' LPE
LP!
LPS LPG LPA TX
CH
Lipid
Fig. 2. Activation of t h e y e a s t plasma m e m b r a n e A T P a s e by different lipids. The activity with soybean lipids (SB), taken as 100%, was 2.6 Aanol • min -1- m g p r o t e i n -1. O t h e r details as in Fig. 1.
121 Table I. Activation of the yeast plasma membrane ATPase by lipids extracted from yeast plasma membranes. 100 ~g lipids w e r e mixed with 10 ~g enzyme as described in Methods. Lipid source
-
Soybean Plasmamembranesfrom glucose-fermentingyeast Plasmamembranesfrom glucose-starvedyeast
Extraction procedure
ATPase activity (umol/min"mg protein-z)
Bligband Dyer Butanol Blighand Dyer Butanol
0.06 1.4 0.8 1.0 0.7 1.1
has been observed with crude phospholipid preparations from such diverse systems as soybean (Fig. 2), yeast plasma membranes, bovine brain and Escherichia coli (data not shown). Therefore, during the expression of the yeast plasma membrane ATPase in other organisms no problems are expected with the lipid composition of the new host membranes. The utilization of lysophospholipids and, in the case of phospholipids, of different reconstitution procedures greatly facilitates the interpretation of lipid specificity studies. In previous work with plant plasma membrane ATPases these precautions were not adopted and contradictory results were obtained. An ATPase preparation from radish (Raphanus sati~ts) seedlings was activated by PI but neither PC nor PE were effective [17]. On the other hand, an enzyme from mung beans (Vigna radiata) was activated by PC, PS and PG but neither PI nor PE were effective [18]. LPC was effective in both preparations but as other lysophospholipids were not tested it is not clear to what extent the observed specificity reflects the reconstitution requirements of these enzyme preparations. A detailed study of the lipid specificity of the plasma membrane ATPase from Schizosaccharomyces pombe [19] indicated that all phospholipid species activated to a similar extent and that saturated and unsaturated LPC were also equivalent. Therefore this enzyme has the same poor lipid specificity as the plant
ATPase. On the other hand, the enzyme from another fungus, Neurospora crassa, seems to require negatively charged phospholipids [20], resembling the yeast (Saccharomyces) ATPase. From previously studied membrane enzymes, the (Na/K) ATPase from animal plasma membranes exhibits the same requirements for negatively charged polar heads and unsaturated hydrophobie chains as the yeast ATPase [21,22]. On the other hand, the (Ca) ATPase from sarcoplasmic reticulum can be activated by detergents such as Triton X-100 and Brij 52 [23] and by all phospholipid species, although with slight preference for PC [24]. Therefore this latter enzyme has a similar lipid specificity as the plant ATPase. These results and the experiments performed with lipids extracted from yeast plasma membranes make it unlikely that the activation of the yeast and plant ATPases by growthpromoting factors [ 3 - - 5 ] were mediated by the generation of an activating lipid. Recent reports on lipid changes induced by glucose in yeast [25] and by auxins in plants [26,27] may be related to the activation of other proteins connected with the growth response. Acknowledgements This work was partially financed by a grant of the Spanish Comision Asesora de Investigacion Cientifica y Teeniea. J.S. was a fellow of the Fundacion March.
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