Lipid-binding proteins in plants

Otemisto' and Ph.vsics of Lipids. 38 (1985) 51-62 Elsevier Scientific Publishers Ireland Ltd.

51

LIPID-BINDING PROTEINS IN PLANTS

JEAN-CLAUDE KADER Laboratoire de Physiologie Cellulaire, U.A.C.N.R.S. 566, Universit~ Pierre et Marie Curie, 4 place Jussieu, 75005 Paris {France)

Received April 9th, 1985 Plant cells contain water-soluble proteins able to facilitate in vitro intermembrane transfer of phospholipids (phospholipid transfer proteins, PLTP) or to bind long-chain fatty acids (fatty acid binding proteins, FABP). PLTPs, purified to homogeneity from various plant organs (seeds or leaves) are, in major part, low molecular-mass proteins (9 kDa), basic (pI around 9) and non-specific for phospholipids. FABPs, purified from seedlings, have also a low molecular mass (8.7 kDa), a high pl (8.4) and are non-specific for fatty acids. Interestingly, recent observations indicated that PLTPs are also able to bind fatty acids. Are PLTPs and FABPs the same protein? To try for an answer to this question, a comparison of the properties of both categories of proteins will be presented in this paper. Also, possible physiological functions of these proteins will be considered and hypotheses on their role in vivo will be discussed. In addition, new perspectives opened by the use of these proteins as tools for membrane studies will be presented. Key words: phospholipid; fatty acid; transfer; binding; proteins; plants.

1. Introduction The first indications that intermembrane lipid exchange occurs in plant cells have been given by in vitro experiments carried out in 1970 with labeled phospholipid-containing microsomes and unlabeled mitochondria [1]. These experiments showed that soluble protein extracts from various plants were able to facilitate these lipid inovements. However, the isolation of purified P L T P , defined by their ability to mediate in vitro intermembrane exchange of phospholipids, was hampered by the presence of lipases and proteases in crude cytosols. The isolation of homogeneous PLTPs was successfully obtained only in the last three years from seeds [2,3] or green leaves [4]. In contrast, pure PLTPs fiom animal tissues have been

Abbrevations: DPG, diphosphatidylglycerol; FABP, fatty acid binding protein; ns-LTP, non specific lipid transfer protein; MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PC-TP, phosphatidylcholine-transfer protein; PE, phosphatidylethanolamine; PG, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PI-TP, phosphatidylinositol transfer protein; PLTP, phospholipid transfer protein. 0009-3084/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

52 purified more than ten years ago (see Ref. 5 for review). The same lag in the knowledge was observed for proteins able to bind long-chain fatty acids and oleoylCoA (fatty acid-binding proteins, FABP) well studied in animal cells (see reviews in this volume) and only recently purified from plant cells [6]. Interestingly, il appeared that a plant PLTP (from spinach leaves) also possesses binding ability for fatty acids {7]. From this observation, rose the possibility that PLTPs and FABI's are identical proteins. It is the aim of this paper to compare the properties of these proteins and to discuss their physRilogical significance in plant cells.

2. Lipid Transfer and Binding Assays A. LipM transfer assay The activity of PLTP was determined by following the transfer of labeled phospholipids between natural membranes such as microsomes and mitochondria [8] or, more often, between artificial (liposomes) and natural (mitochondria) membranes [2]. Routine assays were carried out with liposomes made from [3H]phosphatidylcholine; cholesteryl [IJ4C]oleate or glycerol[~'~C]trioleate were added as non-exchangeable tracers since they are nol transferred by plant PLTPs; the same is true for animal proteins [5))]. The doubly labeled liposomes were incubated with unlabeled mitochondria and appropriate amounts of crude cytosolic proteins or purified PLTPs. After centrifugation, tire determination or ~H-and va(,_ labels recovered in mitochondrial pellets allowed the calculation of tire rate or transfer of [3H]PC corrected for cross-contamination ot milochondria by intact liposome~. Other transfer assays involved two categories of liposomes, one conraining a glycolipid. After incubation in the presence of PLTP, glycolipid-contammg liposomes were agglutinated by addition of leclin [10.11J. Multilamellar vesicles have also been used as receptor membranes for transfer assays with planl PLTP (Guerbette, unpublished). Lipid transfer can also be [\)llowed continuously by using liposomes containing spin-labeled phospholipid; ESR spectra clearly showed the movement of lhis phospholipid towards unlabeled liposomes m the presence of various plant cytosolic extracts [ l l J . B. l, ipid binding assa.v Various methods are available to detect FABPs. After incubation of protein with labeled fatty acid (generally [I J4( TM]oleic acid), Ihe fatty acid-protein complex w.as isolated by gel filtration or ion exchange chromalography and detecled by radioactivity measurements J6]. Alternatively, isoelectric focusing was used to separate fatty acid-protein complexes from unbound lipid: autoradiography, in parallel with protein staining, allowed the detection of the labeled complexes [6]. Binding kinetics were studied by a dextran-coaled charcoal method row)lying lhe separation of bound ligand (fatty acid) from unbound ligand by precipilaling the latter with the adsorbent [6].

53 3. P u r i f i c a t i o n P u r i f i c a t i o n o f PLTPs and F A B P s From plant cells, k n o w n to be rich in p h e n o l i c c o m p o u n d s , lipases and proteases w h i c h inactivate e n z y m e s , n e e d e d t e c h n i q u e s a d a p t e d to these materials.

0.2

0.3 02

,,," • t. ~--~ o CM1 I ~ ,=

/

| 0.1

..

~.'>, .,,

I0

0.1

,;... j o

0 0

20

40

60

80 f r a c t i o n s _ - - _

1 o-~B ,.. CMI oo.

,

0.4. 0.2.

R C

A

oo

r,,,T 1L J';-'[.

CMIIIo° ~.

~"

40 "

20

f

0

200

400

ml

Fig. 1. Purification of PLTP from spinach leaf (A) and FABP from oat seedlings (B), A: active proteins from spinach leaves were chromatographed on a CM-Sepharose column; a gradient of sodium phosphate (C in M, open circles) eluted three fractions transferring PC between liposomes and mitochondria fR, expressed as % of PC transferred, closed circles). Absorbance at 280 nm is indicated by dashed line. The experimental details are given in Ref, 4. B: after incubation with [ l-~'C]oleic acid, proteins from oat seedlings were chromatographed on a CMSephadex column; a tractions (R, expressed as thousands of cpm, closed circles). Absorbance at 280 nm is indicated by dashed line. The experimental details are given in Ref. 6,

54 A. t'L TPs

These proteins have been purified from plant cytosols by successive gel filtration and ion-exchange chromatography. In typical experiments, PLTPs from spinach leaves were purified from cytosolic extracts by chromatography on Sephadex G-75 [4]. The low molecular mass-fractions, containing phospholipid transfer activity (determined by liposome-mitochondria assays) were pooled and chromatographed on a DEAE-Sepharose column; the major part of the activity was associated with unbound fractions corresponding to basic proteins. Only a minor proportion of the activity, corresponding to acidic proteins, was eluted by a gradient of NaCI. The basic proteins were then chromatographed on a CM-Sepharose column. Three main peaks of activity were detected (CM0, CMI, CM2) (Fig. 1). The CM2 fraction exhibited only one band after SDS-polyacrylamide gel electrophoresis, with an apparent molecular mass of 9 kDa. The purity of this fraction was confirmed by high-performance liquid chromatography on reverse-phase column (data not shown). The 'CM2' band, accompanied by other ones, was also found in CM0 and CM1, suggesting that these fractions contained isoforms of CM2 protein. Similar results have been found for maize proteins [2]. Interestingly, isoforms have been described for animal ns-LTP [12]. B. FABPs

The first FABP isolated from plant cells was obtained from oat seedlings [6]. Cytosolic proteins, after a heat-treatment to eliminate proteins, were chromatographed on Sephacryl S-200. The low molecular mass-fractions, containing the lipid-binding proteins (detected as [14C]oleic acid-labeled fractions)were pooled and chromatographed on a CM-Sephadex column. Three main peaks (CMI, CMI1, CMIlI) of lipid-binding activity were detected (Fig. 1). The CMI fraction, after delipidation, was purified by preparative isoelectric focusing. The major band, focusing at pH 8.4, was recovered as the pure fatty acid binding protein (9 mg of protein from 500 g of etiolated seedlings).

4. Properties The availability of purified PLTPs and FABPs from plants allowed detailed studies of their properties. A. Molecular mass

A remarkable homogeneity was noted for the molecular mass determined by SDS-electrophoresis or gel filtration for basic PLTPs purified from higher plants (Table 1). Starting from maize [13], castor bean [3] or barley (Douady, unpublished) seedlings, pure PLTPs were obtained with molecular mass of 9 kDa, identical to the value found for spinach leaf PLTP ('CM2 fraction'). However, dimeric forms of maize PLTP have been observed after SDS-electrophoresis carried out in non-reducing conditions, giving an apparent molecular mass of 20 kDa [2] ;

55 TABLE l PROPERTIES OF PLTPs AND FABPs FROM HIGHER PLANTS Source

Molecular mass (kDa)

pl

Lipid transferred

Ref.

2 13 4 7 Douady, unpublished 3

PL TPs Maize seedlings Maize seedlings Spinach leaves

20 a 9a 9a

8.8 8.8 9

Barley seedlings

9a

>7.5

PC, PE, PI PC, PE, PI PC, PE, PI, PG Fatty acids, acyl-CoA PC, PE, PI

Castor bean seeds

9a

>7.5

PC, PE, PI, PG, MGDG

FABP Oat seedlings

8.7 a

8.4

All straight-chain fatty acids oleoyl and palmitoyl-CoA

6

aDetermined by SDS-electrophoresis. bDetermined by gel filtration. dimeric and trimeric forms of spinach PLTP have been detected (Kader, unpublished). The value found in average for a basic plant PLTP is rather low when compared to those determined for other PLTPs. The values vary from 11.2 kDa for rat hepatoma ns-LTP to 24.6 kDa for bovine liver PC-TP and 32.3 kDa for rat brain P1-TP (see Ref. 1 and 5 for reviews). Bovine liver ns-LTP, which is a basic protein, has a molecular mass of 14.5 kDa. In addition to these pure basic proteins, other PLTPs have been only partially purified from potato tubers (the first PLTP detected in plants) [14], cauliflower florets [1] and oat leaves [11]. Plant cells also contain acidic PLTPs which have been well studied in castor bean seedlings. Various PLTPs have been found with molecular mass varying from I1.1 to 69.2 kDa [15]. Interestingly, acidic PLTPs have been highly or partially purified fiom yeasts, lhngi or bacteria. PLTP from Saccharomyces cerevisiae has a molecular mass of 35 kDa [16] and PLTP [17] from Rhodopseudomonas sphaeroides, 27 kDa, whereas a value o f 20 kDa was found for a partially purified PLTP from Mucor mucedo [ 18]. A molecular mass of 8.7 kDa was determined for FABP from oat seedlings as shown by SDS-urea-electrophoresis [6]. This value is very close to that (9 kDa) found for basic PLTP from plant. This molecular mass is lower than that (12 kDa) determined for rat liver FABP [19].

B. Isoelectric point PLTPs from plant tissues, studied so far, are in major part basic with pl around 9 for spinach and maize proteins (Table I). The same is true for other seeds studied

56 (barley and castor bean). In addition, PLTPs with low pl have been detected in spinach leaf cytosols, whereas pI values varying from 5.4 to 6.6 have been attributed to castor bean acidic PLTP [15]. The occurrence of both basic and acidic PLTPs is well established in animal cells. Values around 5 have been found for pI of rat hepatoma ns-LTP, bovine and rat liver PI-TP; pl is equal to 5.8 for bovine liver PC-TP, whereas rat liver PC-TP and ns-LTP have a high pI (8.4 and 9), respectively (see Refs. 1 and 5 for reviews). Bovine liver ns-LTP has a pl around 9.5 [ 12], remarkably close to that found for several basic plant PLTPs. As indicated above, oat FABP is a basic protein, as shown by its pI (8.4) determined by isoelectric focusing [6]. However, this result is only valid when the protein is delipidated with butanol before focusing. When the protein is lipidated by incubation with fatty acid, a shift of its pl from 8.4 to 4.8 was observed, due to the formation of the lipid-protein complex. It has been suggested that this pl shift is due to conformational changes in the protein creating new negative surface charges [6]. C. Stability Both PLTPs and FABPs are remarkably stable. Maize PLTP and oat FABP keep their activity after heating [2,6] ; for this reason a heat treatment has been included in the purification procedures of both proteins [6,13]. Oat FABP, after lyophilization, could be stored at --20°C for several months without loss of binding activity [6]. The activity of spinach of maize PLTP only slightly decreases (10% per month) when the solutions are stored at +4°C, at a concentration lower than 1 mg • ml -~ ; in another method of storage, the solutions were dialyzed against 50% glycerol (w/v) and kept at -20°C. Alternatively, PLTPs can be lyophilized and kept at --20°C. This stable activity as well as the high yield (about 30 mg of PLTP starting from 4 kg of maize seeds, for instance) facilitated the characterization of these proteins. D. Amino acid composition and primary structure The amino acid compositions of maize and spinach PLTP are characterized by the absence of phenylalanine, whereas glycine and alanine are major compounds (Table II) [30]; the presence of cysteine in both proteins could explain the formation of polymers in non-reducing conditions. Cysteine is also abundant in oat FABP (Rickers and Spener, pers. comm.) (Table 11); glycine, alanine and aspartic acid (asparagine) are the major amino acids. A comparison of these compositions with those recently determined for ns-LTP from bovine liver [21], in agreement with previous analysis [ 12] indicated that this protein, comprising 121 amino acids, lacks histidine, arginine and tyrosine and contains one cysteine and one tryptophan residue. This composition was based on the primary structure of the protein which is now completely elucidated [21]. Serine and alanine are the amino and carboxy terminal residues respectively. For spinach PLTP, only a partial primary structure has been described (34 amino acids from amino terminal end); glycine is

57 TABLE 1I AMINO ACID COMPOSITION OF NON-SPECIFIC PHOSPHOLIPID TRANSFER PROTEINS AND FATTY ACID BINDING PROTEIN Amino acid

Asx Thr Ser Glx Pro Gly Ala Val Cys lle Leu Tyr Lys Arg Met Phe Trp His Total

PLTP from maize seeds

PLTP from spinach leaves

ns-LTP from bovine liver

1131

[20]

[211

12 6 16 3 4 12 18 5 3 6 4 2 3 5 1 0 ndb 1

8 6 8 2 6 14 14 4 6 7 6 2 6 1 2 0 nd b 2

15 5 6 12 4 14 9 8 1 5 11 0 18 0 5 7 1 0

9 5 5 6 4 9 8 5 4 3 4 3 4 4 1 3 1 3

121

81

101

94

FABP from oat seedlingsa

aRickers and Spener, pers. comm. bNot determined.

the amino terminal and histidine, the carboxy-terminal residues [20]. From the comparison of primary structures of bovine and spinach PLTPs, it was found that two regions at least presented homology (Fig. 2). This comparison could reveal other interesting points when primary structures of plant PLTPs and also plant FABPs will be elucidated.

5. Specificity A property shared in common with all basic PLTPs purified to homogeneity from various plant cells is their broad specificity for transferring phospholipids. In addition to PC, other phospholipids - PI, PC and to a lesser extent PE - are transferred by spinach leaf PLTP [4]. In the same tissue, a cytosolic protein, with a molecular mass of 11 kDa has been recently detected; in addition to an ability to transfer PC, this protein also transferred spin-labeled MGDG. This galactolipid transfer protein could be identical to the spinach leaf PLTP [22].

58

31

i, 3~

33

34

35

36

A

I le~ I¥-G ly- I letPh e-r~ ]

B

Ser~ly-Gly

30 [31

A e

32

33 / ~" I ~

I

- lie .JLys -J AI a J

42

43

44

45

46

47

24

| 23

22

21

20

[ 19

GlytPro-Gly-GIy-LystGlu k euIPro.Gly-Gly" k ys~L ~ eu

Fig. 2. Homologies between the primary structures of bovine liver non-specific phospholipid transfer protein from bovine liver (A, Ref. 21) and phospholipid transfer protein from spinach leaf (B, Ref. 20).

Basic PLTPs from plants resemble non-specific proteins from animals which are able to transfer all common phospholipids (except DPG) [5]. Animal ns-LTP can also transfer cholesterol; it is now accepted that these proteins are identical to sterol carrier protein 2 [23,24]. The eventual participation of plant PLTPs in a similar transport of sterolic compounds remains to be established. In addition to these basic non-specific plant PLTPs, proteins from castor bean seeds have been reported to transfer specifically PC or PI [15]. It is of interest to note that plant FABP exhibits a broad specificity for fatty acids, regardless of their degree of unsaturation [6].

6. Binding Properties and Mechanisms of Action Interactions between lipids and proteins are necessarily involved in the activity of PLTPs and FABPs. Several informations are available now on the binding of lipids by these proteins. A. PL TPs

It has been established that bovine liver PC-TP binds specifically 1 tool PC/mol protein [5]. One could expect that plant PLTPs are also able to bind various lipids since these proteins are non-specific. Indeed, binding experiments performed with maize protein revealed that when [3H]PC-containing liposomes were incubated with maize PLTP, labeled protein-lipid complexes were obtained by chromatofocusing [2]. However, only a weak percentage of the initial label was

59 recovered in the protein. This weak binding of PC was confirmed by analytical isoelectric focusing with spinach PLTP [7]. Moreover, maize PLTP as well as spinach PLTP - or also bovine liver ns-LTP [12] do not contain endogenous phospholipids when isolated. From these observations, questions arose about the mode of action of planl PLTPs. It is unlikely that they act in a manner similar io bovine liver PC-TP by exchanging their bound PC with PC molecules from membranes: Ibis shuttle mechanisms allows PC-TP to exchange PC from one membrane to another. Net transfer is observed when PC-TP leaves a membrane without PC bound [5]. Animal nonspecific transfer protein may act in a different manner as recently suggested [25]: these proteins may accelerate the spontaneous exchange of lipid monomers between membranes by lowering tire energy barrier to dissociation and association of lipid monomers to membranes. We suggest that a similar mechanism works for plant PLTPs, partly explaining their broad specificity for lipids. An important point is to know whether plant PLTPs facilitate an exchange or net transfer of lipids between membranes. Exchange of phospholipids was observed in transfer activity assays like labeled liposome-unlabeled milochondria, since the increase in specific radioactivity of mitochondrial lipids is accompanied by a parallel decrease in specific radioactivity of liposomal lipids; this decrease is due to a back transfer lipids from mitochondria to liposomes. In addition to exchange, a net transfer process, leading to an increase in the mass of lipids of mitochondrial membranes was recently observed with maize PLTP (Guerbelte, unpublished). It is now well established that ns-LTP fl-om bovine liver mediates a net mass transfer of lipids [26,27]. It can be concluded lhat animal and plan! non-specific lipid transfer proteins facilitate both exchange and net transfer of lipids between membranes: the relative importance of these two processes probably depends on the lipid composition of the membrane, on the ionic environmenl and on the relative amount of the two membrane lipid pools. B. FABPs Oat FABP is able to bind all straigh|-chain fatty acids from Ci6 to C2o, saturated or unsaturated as well as oleoyI-CoA and pahnitoyl-CoA [6]. However, the protein does not exhibit binding ability for PC. Recent observations indicated that pure spinach leaf PLTP behaved as a FABP, since a tight binding of oleic acid or oleoyl-CoA was obsepced after isoelectric tk)cusing [7]. However, titration curves did nol indicate the p[ shift as observed for oat FABP. in these experiments, it was checked lhal PLTP kepl its transferring activity for phospholipids. It is now necessary to give correct interpretations of these new observations. Do plant PLTPs and FABPs belong to a class of proteins with common tasks? What is the link between fatty acid binding and phospholipid transfer property? Additional information on these proteins is needed to answer these questions.

60

7. Physiological Roles PLTPs and FABPs, by interacting with lipids and membranes, may play important roles in the intracellular dynamics of lipids. A. Role in membrane biogenesis Since PLTPs can convey phospholipids between membranes, it is tempting to suggest that these proteins participate in membrane biogenesis and renewal. PLTPs could carry phospholipids from their sites of biosynthesis, for example PC and PE from the endoplasmic reticulum [28] towards membranes unable to form these lipids; it has been shown that mitochondria [28] or chloroplast envelope [29], although containing PC, do not synthesize this lipid. The observations that spinach PLTP transfer PC towards mitochondria or intact chloroplasts [4] as well as towards chloroplast envelope [30] gave arguments in favor of this hypothesis. An interesting question concerns the eventual presence of PLTP within the stroma of chloroplasts; no transfer activity for PC was detected in the stroma of spinach chloroplasts [31] whereas a galactolipid transfer activity has been recently found [22]. The concept of a participation of PLTP in membrane biogenesis has also been developed for animal cells [32]. However, a clear demonstration of the operation of PLTP in vivo is still lacking. If it is accepted that lipid movements occur between cell compartments, other mechanisms could be suggested, in addition to PLTPmediated movements: spontaneous diffusion of lipids and transfer of membrane vesicles that fuse with other membranes ('membrane flow' theory) [33]. However, the spontaneous diffusion is too slow to be of physiological significance when common phospholipids are considered [25], whereas the membrane flow theory is unable to explain the differences in lipid composition observed in the various cell membranes. B. Role in lipid metabolism Various roles may be attributed to PLTPs and FABPs in lipid metabolism of plant cells. It has been suggested that PLTPs are involved in a cooperative scheme associating chloroplast and endoplasmic reticulum for the biosynthesis of galactolipids (MGDG having 2 linolenoyl moieties) [34,35] ; according to this hypothesis, linoleoyl-PC formed in endoplasmic reticulum (containing oleate-desaturase activity) is transferred towards chloroplasts (containing linoleate-desaturase activity) by a PLTP-mediated process. From linoleoyl-PC transferred, diacylglycerols are t'ornled, allowing the biosynthesis of dilinolenoyl-MGDG. A biosynthesis of dilinolenoylMGDG has been indeed observed when oat plastids were incubated with dilinoleoylPC-liposomes and castor bean plastids [36]. Plant FABPs presumably play important roles in fatty acid metabolism by regulating the intracellular level of free fatty acids and their CoA-esters. The fact that PLTPs, like the spinach protein, bind long-chain fatty acids can explain results obtained in reconstitution experiments comprising spinach chloroplasts, microsomes and spinach PLTP, in the presence of [1-~4C] acetate [35] ; the observed increase in

61

the level of fatty acid-labeling could be due to a direct effect of spinach PLTP on fatty acids rather than on phospholipids. 8. Perspectives for Future Researeh

Since plant PLTPs and FABPS have been recently purified, informations concerning their biochemical properties and their structure have to be completed. In particular, it is necessary to compare PLTP and FABP from the same plant in order to evaluate their protein identity as suggested by the experiments with spinach PLTP [71. The mode of action of these proteins is still unknown and much has to be done tbr analyzing the interactions between PLTP and FABP and membrane lipids and for studying the site of binding of fatty acids or other lipids. Other important aspects are the effects of the lipid composition of membranes involved in exchange processes and the influence of ionic environment (see Refs. 5 and 37 for reviews). These studies could demonstrate the importance of the surface charges of membranes on the activity of PLTPs as suggested by the inhibiting effect of phosphate on maize PLTP [2]. In order to establish the physiological function of these proteins, several approaches have to be developed: determination of an eventual correlation between the amount of proteins (determined by immunochemical techniques or highperformance liquid chromatography), study of the biogenesis of these proteins by using techniques of molecular biology, study of the cellular organization of lipidbinding proteins, probably partly linked to membranes. FABPs and PLTPs may play regulatory roles on the activity of membranebound enzymes, in particular, by transporting water-insolube substrates towards active sites of membraneous enzymes; these eventual roles have to be explored. An important aspect for future research is to consider the possible use of these proteins to study lipid-protein interactions within membranes (transbilayer movement, asymmetry of lipid distribution). Another interesting field of investigation is to examine the possibility to manipulate the membrane lipid composition by incubating menlbranes with PLTP and liposomes of controlled composition. The eventual effects of qualitative and quantitative changes in phospholipid or fatty acid composition on structure and function of membranes could {}pen promising perspectives for a better understanding of the organization of plant cell membranes. References 1 J.C. Kader, in: G. Poste and G.L. Nicolson (Eds.), Cell Surface Review, Vol. 3, Elsevier/ North-Holland, Amsterdam, 1977, pp. 127-204. 2 D. Douady, M. Grosbois, F. Guerbette and J.C. Kader, Biochim. Biophys. Acta, 710 (1982) 143-153. 3 M.Yamada, I. Nishida, S.I. Watenabe and J.C. Kader, in: P.A. Siegenthaler and W. Eichenberger (Eds.), Structure, Function and Metabolism of Plant Lipids, Elsevier/North-HoUand, Amsterdam, 1984, pp. 291-294.

62 4 J.C. Kader, M. JulienneandC. Vergnolle, l,ur.J. Biochem., 139 (1984) 411 416. 5 K.W.A. Wirtz, in: P.C. Jost and O.H. Griffiths (Eds.), Lipid-Protein Interactions, Wiley Interscience, New York, 1982, pp. 151-233. 6 J. Rickers, I. Tober and F. Spener, Biochim, Biophys. Acta, 794 (19841 313 319. 7 J. Rickers, F. Spener and J.C. Kader, FEBS Lett., 180 (19851 29-32. 8 T. Tanaka and M. Yamada, in: L.A. Appelqvist and C. Liljenberg (Eds.), Advances in the Biochemistry and Physiology of Plant Lipids, l-lsevier/North-Holland, Amsterdam, 1979, pp. 243-248. 9 D.B. Zilversmit and M.E. Hughes, Methods Membrane Biol., 7 (1976) 211 259. 10 F. Guerbette, D. Douady, M. Grosbois and J.C. Kader, Physiol. Vdg., 19 (19811 467 472. 11 T. Tanaka, J.l. Ohnishi and M. Yamada, Biochem. Biophys. Res. Commun., 96 (1980) 394-399. 12 R.C. Crain and D.B. Zilversmit, Biochemistry, 19 (1980) 1433-1439. 13 D. Douady, M. Grosbois, 1:. Guerbette and J.C. Kader, Physiol. V6g., 23 (19851 in press. 14 J.C. Kader, Biochim. Biophys. Acta, 380 (1975) 31-44. 15 T. Tanaka and M. Yamada, in: J.F.G.M. Wintermans and P.J.C. Kuiper (Eds.), Biochemistry and Metabolism of Plant Lipids, Elsevier/North-Holland, Amsterdam, 1982, pp. 99-106. 16 G. Daum and F. Paltauf, Biochim. Biophys. Acta, 794 (1984) 385-391. 17 S.P. Tai and S. Kaplan, J. Biol. Chem., 259 (1984) 12178-12183. 18 H. De Scheemaeker, C. Vergnolle, F. Tchang, L. Chavant and J.C. Kader, in: P.A. Siegenthaler and W. Eichenberger (Eds.), Structure, Function and Metabolism of Plant Lipids, Elsevier/North-Holland, Amsterdam, 1984, pp. 303 306. 19 R.K. Ockner, J.S. Manning and J.P. Kane, J. Biol. Chem., 257 (1982) 7872 7878. 2(/ P. Bovilon, C. Drischel, C. Vergnolle, J.C. Kader and H. Duranton, C.R. Acad. Sci. Paris, 300, Set. 111 (1985) 421. 21 J. Westerman and K.W.A. Wirtz, Biochem. Biophys. Res. ('ommun., 127(19851 333-338. 22 1. Nishida and M. Yamada, Biochim. Biophys. Acta (1985) in press. 23 J.M. Trzaskos and G.L. Gaylor, Biochim. Biophys. Acta, 751 (1983) 5 2 - 6 5 . 24 B.J. Noland, R.E. Arebalo, E. Hansbury and T.J. Scallen, J. Biol. Chem., 255 (1980} 4282-4289. 25 J.W. Nichols and R.E. Pagano, J. Biol. Chem., 258 (1983) 5368-5371. 26 R.C. Crain and D.B. Zilversmit, Biochemistry, 20 (19811 5320-5326. 27 P.F.H. Franck, J.M. De Ree, B. Roelofsen and J.A.F. Op den Kamp, Biochim. Biophys. Acta, 778 (1984) 405-411. 28 T.S. Moore, Plant Physiol., 65 (1980) 1076-1080. 29 J. Joyard and R. Douce, C.R. Hebd. Sdances Acad. Sci. Paris, 282, Set'. D (19761 1515 1518. 30 M. Miquel, M.A. Block, J. Joyard, A.J. Dome, J.P. Dubacq, J.C. Kader and R. Douce, In: P.A. Siegenthaler and W. Eichenberger (Eds.), Structure, Function and Metabolism of Plant Lipids, I.lsevier/North-Holland, Amsterdam, 1984, pp. 295 298. 31 J.P. Schwitzguebel, T.D. Nguycn and P.A. Siegenthaler, in: P.A. Sicgenthaler and W. Eichenberger (Eds.), Structure, Function and Metabolism of Plant Lipids, Elsevier/NorthHolland, Amsterdam, 1984, pp. 299-302. 32 K.W.A. Wirtz, Biochim. Biophys. Acta, 344 (1974) 95 117. 33 D.J. Morrd, Annu. Rev. Plant Physiol., 26 (19751 441 481. 34 D. Drapier, 1.P. Dubacq, A. Tr6molibres, C. Vergnolle, M. Julienne and J.('. Kader, in: J.F.G.M. Wintermans and P.J.C. Kuiper (Eds.), Biochemistry and Metabolism of Plant Lipids, Elsevier/North-Holland, Amsterdam, 1982, pp. 43 46. 35 J.P. Dubacq, D. Drapier, A. Tr~moli~res and J.C. Kader, Plant Cell Physiol., 25 (1984) 1197-1204. 36 J.l. Ohnishiand M. Yamada, Plant Cell Physiol., 23 (1982) 767 773. 37 J.C. Kader, D. Douady and P. Mazliak, in: J.N. Hawthorne and G.B. Ansell (l'ds.), Phospholipids, Elsevier/North-Holland, Amsterdam, 1982, pp. 2 7 9 - 311.