Monogalactosyldiacylglycerol (MGDG), ()the major plastid glycerolipid (Benson, 1964; Douce and Joyard, 1980), is synthesized in plastid envelope membranes (Douce, 1974; Block et al., 1983) by a UDPgalactose:1,2-sn-diacylglycerol 3--D-galactosyltransferase (EC 2.4.1.46), or MGDG synthase. Despite its importance for chloroplast membrane biogenesis, MGDG synthase is only a minor envelope protein (Covès et al., 1987; Joyard and Douce, 1987). This enzyme transfers a galactose from a water-soluble donor, UDP-galactose (UDP-Gal), to a hydrophobic acceptor molecule, 1,2-diacylglycerol (Ferrari and Benson, 1961; Neufeld and Hall, 1964). Teucher and Heinz (1991) and Maréchal et al.(1991) independently purified several hundred-fold MGDG synthase activity from spinach chloroplast envelope. In each case, the final amount of enzyme was so low (in the range of microgram when starting the purification from 0.1-g envelope proteins) that further characterization of the protein or amino acid sequence determination were almost impossible.

Analyses of the kinetic properties of partially purified MGDG synthase (Maréchal et al., 1994a), using mixed micelles containing 1,2-diacylglycerol, CHAPS, and phosphatidylglycerol (PG), demonstrated that the ``surface dilution'' kinetic model proposed by Deems et al.(1975) was valid for MGDG synthase. Maréchal et al. (1994a) showed that MGDG synthase was a sequential, either random or ordered, bireactant system. The affinity of MGDG synthase for each substrate (UDP-Gal and 1,2-diacylglycerol) did not vary when the cosubstrate supply was varied. To investigate the functional independence of substrates binding within MGDG synthase active site, we undertook an analysis of the enzyme structure using inhibitors and protection of the enzyme by its substrates. In this study, we distinguish inactivation of MGDG synthase, due to a modification of enzyme structure (such as a covalent linkage with an amino acid reagent or the extraction of a protein-associated metal with a chelating agent) from inhibition, due to the binding of a substrate analogue.

EXPERIMENTAL PROCEDURES

Materials

Purification of Spinach Chloroplasts Envelope Membranes

Solubilization and Partial Purification of MGDG Synthase

Assay of MGDG Synthase

Incubation Conditions

Inactivation Kinetic by Metal Chelators

Radiolabeling with [S]SLR

Protein Determination

RESULTS AND DISCUSSION

MGDG Synthase Inhibition by UDP

Figure 1: MGDG synthase inhibition by UDP. The representation is according to Lineweaver and Burk. Initial velocities of MGDG synthase (corresponding to 7 µg of protein) activity were determined as described under ``Experimental Procedures.'' A, UDP-Galactose is the varied substrate, dioleoylglycerol mol fraction is kept constant (0.03 mol fraction). The family of curves exhibits a competitive inhibition pattern with K = 23.5 ± 1.5 µM. B, dioleoylglycerol is the varied substrate, and UDP-Gal concentration is kept constant (1 mM). 1/dioleoylglycerol is expressed in mol fraction according to the surface dilution model. MGDG synthase inhibition by UDP shows a noncompetitive pattern that calculated K for UDP to be 52 ± 16 µM. K were determined according to equations from Segel (1975).

These results demonstrate that UDP binds competitively to the UDP-Gal binding site of MGDG synthase, while it does not interfere with the fixation of the second substrate, 1,2-diacylglycerol, in its specific binding site. Thus, they provide additional experimental support for our previous hypothesis (Maréchal et al., 1994a) that the catalytic site of MGDG synthase presents two distinct binding sites, i.e. one for each substrate. MGDG synthase thus presents some similarities with other galactosyltransferases: (a) UDP is in general a competitive inhibitor for glycosyltransferases relatively to their UDP-sugar donors (Beyer et al., 1981; Doering et al., 1989) and (b) enzymes such as the UDP-Gal:3--N-acetylglucosamine 4--galactosyltransferase (EC 2.4.1.38) also possesses two distinct substrate binding sites (Yadav and Brew, 1990, 1991). However, and in contrast with what we observed (Maréchal et al., 1994a), binding of one substrate increased the affinity of the enzyme for its cosubstrate (Barker et al., 1972; Powells and Brew, 1976). This major difference between MGDG synthase and the other galactosyltransferases is not really surprising since one of the two substrates for MGDG synthase, diacylglycerol, is highly hydrophobic.

Comparison of MGDG synthase with 1,2-diacylglycerol-binding proteins is most interesting. For instance, enzymes from the protein kinase C family possessing a 1,2-diacylglycerol regulatory domain and all eukaryotic 1,2-diacylglycerol kinases, contain 1,2-diacylglycerol-binding domains that have very high homology (Sakane et al., 1990; Schaap et al., 1990). As we have demonstrated for MGDG synthase, kinetic analyses of diacylglycerol kinases showed a functional independence of cosubstrates (1,2-diacylglycerol and ATP) binding (Kanoh and Ohno, 1981; Bishop et al., 1986; Wissing and Wagner, 1992). Amino acid sequence analyses of porcine diacylglycerol kinase (Sakane et al., 1990) and human diacylglycerol kinase (Schaap et al., 1990) also suggest that 1,2-diacylglycerol binds to a domain distinct from that of ATP. Moreover, the 1,2-diacylglycerol-binding site of MGDG synthase (Maréchal et al., 1994a, 1994b), like that of protein kinase Cs (Hannun et al., 1991; Azzi et al., 1992) and diacylglycerol kinases (Bishop et al., 1986), has a specificity for 1,2-diacylglycerol molecules that differs according to fatty acid composition. However, similarity of the 1,2-diacylglycerol binding site between all of these enzymes is not complete since phorbol esters do not inhibit MGDG synthase (data not shown) nor diacylglycerol kinases (Sakane et al., 1990). In contrast, phorbol esters bind to the 1,2-diacylglycerol binding site from most protein kinase C (Nishizuka, 1989).

These observations prompted us to further characterize the two substrate binding sites of MGDG synthase. We therefore decided to investigate their structure by using specific reagents for various amino acids. We first investigated whether free amino groups were associated with substrate-binding sites.

MGDG Synthase Inactivation by Citraconic Anhydride

Table 1led to several other conclusions. First, the binding of each substrate, UDP-Gal or 1-2,diacylglycerol, on MGDG synthase is possible in absence of the other one. This is important to understand the enzyme mechanism; since MGDG synthase inactivation by citraconic anhydride was prevented after preincubation with either substrate (Table 1), substrate binding to the enzyme is therefore likely to be random. Second, the marked sensitivity of MGDG synthase to citraconic anhydride clearly demonstrate that primary amino groups do exist in the vicinity of both substrate binding sites. Interestingly, the presence of lysine residues in the UDP-Gal binding site was also demonstrated in animal galactosyltransferases (Yadav and Brew, 1990) and might be essential for catalysis.

Because addition of the enzyme substrates prior to addition of lysine-blocking reagent prevented MGDG synthase inactivation, we decided to use amino-group labeling reagents to characterize the envelope proteins that could be specifically protected from labeling by the addition of UDP-Gal or 1,2-diacylglycerol.

Labeling of MGDG Synthase Inactivation by [S]SLR

MGDG synthase fractions were incubated for 1 h with SLR, (following a preincubation in presence or absence of the enzyme substrates) and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The protein pattern of the different fractions and their labeling are presented respectively in Fig. 2, A and B. As expected because of the low selectivity of SLR, almost each protein present in the fraction was labeled (Fig. 2). However, some polypeptides were relatively more intensively labeled by SLR than others, compared with their Coomassie Blue staining, probably because of a higher amount of lysine residues exposed to the blocking agent. For instance, two polypeptides having a molecular mass of 21 and 22 kDa were rather strongly labeled with S, but only barely detectable after Coommassie Blue staining (Fig. 2). In fact, one should keep in mind that not all proteins are equally labeled: yields of 10-50% labeling are typically obtained with SLR, depending on the content of amino groups accessible to SLR in the protein and the molar ratio of protein to SLR (technical note for SLR, Amersham Corp.).

Figure 2: Differential radiolabeling of partially purified MGDG synthase by SLR. Aliquots (40 µg protein) of a partially purified MGDG synthase fraction (see ``Experimental Procedures'') were incubated for 1 h at 25 °C with 1.3 mM PG with no additive (lane1), with 1 mM UDP-Gal (lane2), or with 80 µM 1,2-dioleoylglycerol (lane3) or 80 µM 1,2-dioleoylglycerol, and 1 mM UDP-Gal (lane4). Each fraction was subsequently incubated with 25 µl of [S]SLR (1 MBq) for 1 h at 25 °C under gentle agitation. Envelope proteins, extracted as described under ``Experimental Procedures,'' were then subjected to polyacrylamide gel electrophoresis in a 7.5-15% acrylamide gradient in the presence of SDS. After completion of the run, polypeptides were stained with Coomassie Blue (A). The gel was dried and exposed overnight to hyperfilm-max, Amersham Corp. (B). Arrows show polypeptides protected against [S]SLR by 1,2-diacylglycerol.

We observed that preincubation of the partially purified enzyme fraction with 1 mM UDP-Gal, 1 h before adding SLR, had no apparent effect on the labeling pattern (Fig. 2B, lane2). In contrast, when 1,2-dioleoylglycerol was added prior to SLR (Fig. 2B, lanes3 and 4), the intensity of labeling of 3 polypeptides around 20 kDa was remarkably modified (Fig. 2, arrows); the labeling of a 22-kDa polypeptide markedly decreased, whereas labeling of a 21- and a 19.5-kDa polypeptides decreased to a lesser extent. No other specific change in protein labeling due to the 1-h preincubation with 1,2-dioleoylglycerol could be noticed. Since (a) the 21- and 22 kDa-polypeptides are barely colored with Coomassie Blue, (b) the three polypeptides around 20 kDa show differential radiolabeling (in the absence and in the presence of 1, 2-diacylglycerol) with [S]SLR, and (c) their radiolabeling seem to be related (i.e. when the labeling of the 22-kDa polypeptide increases, those of the 21- and 19.5-kDa polypeptides decrease and vice versa), it is therefore possible that these three polypeptides could correspond to the same polypeptide labeled with various amounts of SLR molecules. Indeed, covalent binding of a single t-butoxycarbonyl-L-[S]methionine residue to an -lysine side chain of a protein would increase its molecular mass by about 0.2 kDa and would modify its global charge. Thus, the presence of several accessible amino groups on the protein would allow labeling with several SLR molecules and therefore would result in enough increase of the electrophoretic mobility of a 19 kDa-polypeptide to reach apparent molecular mass of 19.5, 21 and 22 kDa by SDS-polyacrylamide gel electrophoresis. Although an unambiguous characterization of these three polypeptides is still lacking, the observations shown in Fig. 2provide additional evidence for MGDG synthase to be associated with a low molecular mass polypeptide, around 20 kDa, as proposed by Teucher and Heinz(1991) and Maréchal et al.(1991). These results raise the question of how such a low molecular mass polypeptide could be compatible with substrate binding in two separate domains. A possibility is that MGDG synthase could be a multimeric enzyme.

MGDG Synthase Protection by DTT and Inactivation by NEM

Figure 3: MGDG synthase inactivation by NEM. A, NEM supply is in the µM range. B, NEM supply is in the mM range (inset). MGDG synthase activity of envelope vesicles prepared in the absence of DTT was measured at increasing concentrations of NEM (A and B). After envelope solubilization by 6 mM CHAPS, as described under ``Experimental Procedures,'' MGDG synthase activity was measured in the absence of DTT (), and in the presence of 1 mM DTT (), at increasing concentrations of NEM.

We then investigated the availability of free -SH groups in MGDG synthase using NEM, a -SH (-S) blocking agent. Indeed, Fig. 3shows that MGDG synthase activity kept in a medium devoid of DTT was gradually inactivated when NEM concentration was increased, whether the enzyme was embedded in its native envelope membrane or whether it was solubilized (Fig. 3). However, membrane-bound MGDG synthase was far less sensitive to NEM than the solubilized enzyme: Fig. 3, A and B, shows that 5 mM NEM were required to totally inactivate MGDG synthase in envelope membranes, whereas 8 µM NEM was sufficient when the membrane was solubilized (Fig. 3A). Addition of 1 mM DTT to solubilized envelope prevented inactivation of MGDG synthase by NEM (Fig. 3A). These results show that (a) reduced cysteines are essential for MGDG synthase activity and (b) the reduced cysteine residues are located in a hydrophobic area of the enzyme. Cysteine residues are protected by the envelope lipidic leaflets, while they are more exposed to NEM when MGDG synthase was included in mixed micelles.

Partially purified MGDG synthase fractions were then used to probe the presence of free -SH groups in the vicinity of the substrate binding sites. Such enzyme fractions behave almost like solubilized envelope membranes with respect to NEM sensitivity (Table 4) and protection by DTT (see Table 3and Table 4). Table 4demonstrates that preincubation of the enzyme fraction with UDP-Gal (or PG alone) did not affect MGDG synthase inactivation by NEM. Interestingly, galactosyltransferases are also inactivated by NEM, but the enzymes are protected by preincubation in presence of UDP-Gal (Kitchen and Andrews, 1974). Apparently, the free reduced cysteine identified by Yadav and Brew(1990) in the UDP-Gal binding domain of various galactosyltransferases seems not to have any equivalent in the UDP-Gal-binding site of MGDG synthase; -SH groups sensitive to NEM seem to be located in different domains in galactosyltransferases and in MGDG synthase.

In contrast with UDP-Gal, preincubation with 1,2-dioleoylglycerol (and PG) protected MGDG synthase against NEM (Table 4). This result indicates that reduced cysteine residues, accessible to NEM, are located very close to the 1,2-diacylglycerol binding site and are topologically distinct from UDP-Gal binding site. This situation shows some similarity with the 1,2-diacylglycerol domain from protein kinase C, which contain characteristic tandem repeats of cysteine-rich motifs; analyses of the structure (Hubbard et al., 1991) of the amino acid sequence (Hannun et al., 1991) and of targeted mutations (Quest et al., 1994) of protein kinase C, demonstrated that zinc cations from 1,2-diacylglycerol binding site were coordinated to 6 cysteine residues and 2 histidine residues. Similarly, analyses of amino acid sequences of porcine diacylglycerol kinase (Sakane et al., 1990) and of human diacylglycerol kinase (Schaap et al., 1990, Fujikawa et al., 1993) demonstrated the existence of two CysHis domains in their 1,2-diacylglycerol binding site. It is therefore possible that cysteine residues are linked to zinc within the 1,2-diacylglycerol binding site of MGDG synthase. A protein kinase D has been recently characterized that exhibits homology with the regulatory domain of protein kinase C and binds 1,2-diacylglycerol (Valverde et al., 1994). Other proteins that manipulate 1,2-diacylglycerol and also contain a cysteine-rich motif include diacylglycerol kinase (Schaap et al., 1990, Fujikawa et al., 1993). Since reduced cysteines are often involved in metal chelation (Freeman, 1973; Vallee and Auld, 1993), we undertook analyses of the metal ions that could be essential for MGDG synthase activity.

Effect of Zinc and Chelating Agents on MGDG Synthase Activity

We then analyzed the effect of various chelating agents (EDTA, 8-hydroxyquinoline, ortho-phenanthroline) or with phenanthrene (a nonchelating analogue of ortho-phenanthroline). In contrast with EDTA and 8-hydroxyquinoline, ortho-phenanthroline and phenanthrene are hydrophobic compounds (Fig. 4). Table 5shows that among the three chelating agents, only ortho-phenanthroline inhibits MGDG synthase. Incubation with phenanthrene did not alter MGDG synthase activity. Therefore, the inhibition by ortho-phenanthroline is not due to the nonspecific intercalation of this planar molecule in a hydrophobic site of the enzyme (Scrutton, 1973) but to its chelating property (Powers and Harper, 1986). Moreover, incubation with ortho-phenanthroline in the presence of 1 mM ZnCl had no effect on MGDG synthase activity (Table 5), indicating that the soluble metal cation competes with MGDG synthase for ortho-phenanthroline binding.

Figure 4: Structure of the metal chelating agents used (EDTA, 8-hydroxyquinoline, ortho-phenanthroline, phenanthrene). Phenanthrene, a nonchelating analogue of ortho-phenanthroline, has been used as a control for ortho-phenanthroline effect. Ortho-phenanthroline is the most hydrophobic chelating agent, and differs from the others by its strong specificity for zinc and iron and because it does not interact with calcium (Power and Harper, 1986). Elements in boldface characters are involved in metal coordination.

A possible explanation for these results is that MGDG synthase has two metal-binding sites within the hydrophobic core of the enzyme: (a) one site (probably containing histidine residues) that could interact with zinc at pH 7.8, leading to MGDG synthase inhibition and (b) another site that could be strongly associated with a metal. This last domain is not exposed to hydrophilic chelating agents (EDTA and 8-hydroxyquinoline) and requires a very hydrophobic chelating molecule (ortho-phenanthroline) to remove its associated metal.

Fig. 5shows time course inactivation of MGDG synthase by 5 mMortho-phenanthroline at 25 °C in the absence or in the presence of each substrate of the enzyme. When UDP-Gal or PG were added to the incubating medium, inactivation by ortho-phenanthroline was not affected. In contrast, the addition of 1,2-dioleoylglycerol (together with PG) was sufficient to prevent MGDG synthase inactivation by ortho-phenanthroline (Fig. 5). This result demonstrates that the metal associated with the apo-MGDG synthase is located in the vicinity of the 1,2-dioleoylglycerol binding site and not in the vicinity of the UDP-Gal binding site. Such a result fits well with our previous observation (Table 4) that reduced cysteines lie in the vicinity of the 1,2-diacylglycerol binding site and provides some indirect evidence for the occurrence of metal-cysteine clusters within MGDG synthase.

Figure 5: Protection of MGDG synthase against inactivation by ortho-phenanthroline. Purified MGDG synthase, pH 6.0, was incubated for 4 h at 25 °C. Incubations were carried out with 1 mM UDP-Gal (+UDP-Gal), with 160 µM 1,2-dioleoylglycerol solubilized in presence of 1.3 mM PG (+PG + 1,2-DG), and with 5 mMortho-phenanthroline (+O-p). After 1, 2, 3, and 4 h, 200-µl aliquots were removed to measure the remaining activity (see ``Experimental Procedures''). The activity is expressed as a percentage of the initial activity.

Fig. 5also demonstrates that when preincubated in presence of 1,2-diacylglycerol, MGDG synthase is not only protected against inactivation by ortho-phenanthroline, but it is activated. This remarkable effect might be due to the protection of another site reacting with metals, such as the site responsible for MGDG synthase inhibition by Zn or Cu at pH 7.8 (see above). The presence of two metal binding sites, apparently antagonist, is not surprising and has been demonstrated in other metalloenzymes. For instance, inter--trypsin inhibitor, a zinc protein (Steinbuch, 1976), is inhibited by zinc (Salier et al., 1980), and so is another zinc protein, aminoacylase I (Wang et al., 1992; Wu and Tsou, 1993).

The inactivation kinetic of MGDG synthase by ortho-phenanthroline is shown in Fig. 6. MGDG synthase, incubated with 0.1 mMortho-phenanthroline at 4 °C, was assayed every 3 h as described under ``Experimental Procedures.'' Data presented in Fig. 6A did not simply fit second-order kinetic patterns for metal extraction by chelating agents (Dumas et al., 1989; Omburo et al., 1992; Wang et al., 1992). Theoretical curves presented in dashedlines in Fig. 6A and B show that inactivation should follow an exponential decrease leading to total inactivation. In contrast, kinetic of MGDG synthase inactivation follows a complex pattern that could be analyzed as two successive second order events. A first event, characterized by a second order constant k"a = 1175 Mh, occurred during the first 15 h and was followed by another inactivation step with a similar second order constant, k"c = 1083 Mh. Total inactivation did not occur, since 10-20% of the initial activity still remained after 33 h of incubation. These results indicate that MGDG synthase is associated with metal(s) in a complex manner. First, it is possible that more than one population of enzyme do exist, each one differing in the strength of its association with the metal and characterized by each event shown in Fig. 6. Second, it is more likely that MGDG synthase contains at least two metal cations with sequential removal, leading to the two successive inactivation steps presented in Fig. 6. The inactivation kinetic observed with MGDG synthase can be compared with that of other metalloenzymes containing more than one metal cation/enzyme. For instance, alcohol dehydrogenase, which contains two zinc atoms (Drum and Vallee, 1970), is never totally inactivated by any chelating agent (Drum et al., 1969a). Moreover, extraction of zinc from alcohol dehydrogenase proceeds sequentially. A first Zn cation is primarily extracted by ortho-phenanthroline (Drum and Vallee, 1970); this extraction is reversible upon the addition of zinc (Drum et al., 1969b). A second Zn cation is subsequently extracted, and this extraction is irreversible (Drum et al., 1969a). The same holds true for another metalloenzyme, phospholipase C, incubated with ortho-phenanthroline (Little and Otnäss, 1975).

Figure 6: Kinetic of the inhibition of MGDG synthase activity by ortho-phenanthroline. Purified MGDG synthase, with pH adjusted to 6, was incubated as described under ``Experimental Procedures'' for 33 h at 4 °C in the presence of 0.1 mMortho-phenanthroline. At given times, 200-µl aliquots were removed to measure the remaining activity (see ``Experimental Procedures''). Top, the activity is expressed as a percentage of initial activity. A control sample of MGDG synthase to which only water was added () showed little change in activity over the observed period. Inhibition of MGDG synthase activity by ortho-phenanthroline () was analyzed according to Dumas et al.(1989) as described under ``Experimental Procedures,'' and did not exhibit any simple second rate order kinetic. Bottom, logarithmic representation of the activity. Two successive inactivation events seem to occur, a and c, with second order rates: k" a = 1175 Mh and k"c = 1083 Mh. The theoretical curve represents a simple second order kinetic for enzyme inhibition by a single inactivation event.

Attempt to Identify the Metal Cation(s) Associated with Apo-MGDG Synthase

CONCLUSION

MGDG synthase catalyses the transfer of a -galactose from a nucleotidic donor (UDP-Gal) to the sn-3 carbon from the glycerol backbone of 1,2-sn-diacylglycerol. Catalysis involves the galactose moiety of UDP-Gal and the sn-3-hydroxyl group from 1,2-diacylglycerol. In this article, we demonstrate that UDP-Gal binds to MGDG synthase at the level of its nucleotidic side, on a site topologically distinct from that of 1,2-dioleoylglycerol binding. In previous articles, Maréchal et al. (1994a, 1994b) demonstrated that MGDG synthase affinity for 1,2-diacylglycerol was dependent upon acyl chain length (16 or 18 carbons), their position on the glycerol backbone (sn-1 or sn-2), and their level of unsaturation. Therefore, 1,2-diacylglycerol binding on MGDG synthase involves its acyl part. Together, these data demonstrate that MGDG synthase active site contains three distinct parts: two independent substrate-binding sites (for UDP-Gal and 1,2-diacylglycerol) and the catalytic site itself (where galactose transfer occurs). Two possibilities for the relative topology of the substrate-binding sites are presented in Fig. 7; either binding sites are completely separated and catalysis occurs after a deep conformational change (Fig. 7A), or binding sites partly overlap (Fig. 7B). Such systems would function with a random, sequential bireactant mechanism.

Figure 7: UDP-Gal and 1,2-diacylglycerol (1, 2-DAG) binding sites in MGDG active site. A, in this case substrate binding sites are totally separated, catalysis implies a deep conformational change to allow galactose transfer. B, in this case substrate binding sites partly intersect, and domains that bind the nucleotidic part of UDP-Gal (UDP-) and the acyl groups of 1,2-diacylglycerol are topologically distinct.

Despite some similarities (such as the involvement of lysine residues in UDP-Gal binding), MGDG synthase seems not to share the main properties of the eukaryotic galactosyltransferases. This is probably because MGDG synthase is involved in glycerolipid biosynthesis (for a review, see Joyard and Douce(1987)) and therefore manipulates hydrophobic substrate and product. In contrast, comparison of MGDG synthase with 1,2-diacylglycerol-binding proteins that have been extensively studied in animals suggests that some biochemical links might exist between chloroplast envelope MGDG synthase and enzymes from the protein kinase C family and eukaryotic 1,2-diacylglycerol kinase family. Moreover, recent studies (Schmidt-Schultz and Althaus, 1994) report that MGDG (which is a very minor glycerolipid in animal tissues but is a marker for myelination) from oligodendrocytes stimulates protein kinase C. Further investigations are now in progress in order to understand the molecular basis for these puzzling similarities between MGDG synthase and eukaryotic 1,2-diacylglycerol kinases. Finally, since prokaryotic 1,2-diacylglycerol kinases are not rich in cysteine and do not contain any metal (Loomis et al., 1985), the study presented in this paper raises the question of a possible eukaryotic origin of MGDG synthase. In fact, chloroplast envelope MGDG synthase has a prokaryotic counterpart, the monoglucosyldiacylglycerol synthase from cyanobacteria. In cyanobacteria membranes, galactolipids (mostly MGDG) are the major polar lipid constituents, and their biosynthetic pathway (for a review, see Murata and Nishida(1987)) presents only limited differences with that described in the chloroplast envelope (for a review, see Joyard and Douce(1987)). One of them is that MGDG is formed by a monoglucosyldiacylglycerol synthase, which catalyzes the transfer of a glucose molecule from UDP-glucose to 1,2-diacylglycerol to form MGluDG (Murata and Sato, 1983), followed by an epimerization of the glucose moiety to galactose. Although, the difference concerns the UDP-sugar and not the 1,2-diacylglycerol binding site, it would be most interesting to compare the properties of MGluDG synthase from cyanobacteria with those of the chloroplast envelope MGDG synthase. In addition, chloroplast envelope membranes contains several other enzymes that manipulate 1,2-diacylglycerol, namely the galactolipid:galactolipid galactosyltransferase, the phosphatidate phosphatase, and the sulfolipid synthase (for a review, see Joyard and Douce(1987)). An intriguing question is to understand whether a common structure for 1,2-diacylglycerol-binding sites from all these proteins do exist.

FOOTNOTES

*

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: DBMS-PCV, CENG, 38054 Grenoble-cedex 9, France. Tel.: 33-76-88-41-84; Fax: 33-76-88-50-91.

()

The abbreviations used are: MGDG, monogalactosyldiacylglycerol; PG, phosphatidylglycerol; DTT, dithiothreitol; NEM, N-ethylmaleimide; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid.

ACKNOWLEDGEMENTS

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