Proc. Nati. Acad. Sci. USA Vol. 87, pp. 71-74, January 1990 Do thylakoids really contain phosphatidylcholine? (/envelope membranes/phospholipids/Spinacia oleraeea) ALBERT-JEAN DORNE, JACQUES JOYARD, AND ROLAND DoUCE* Laboratoire de Physiologie Cellulaire Vdgdtale, Unitd Associde au Centre National de la Recherche Scientifique n' 576, Ddpartement de Recherche Fondamentale, Centre d'Etudes Nucldaires de et Universitd Joseph Fourier, 85 X, F-38041 Grenoble-cedex, France Communicated by A. A. Benson, September 18, 1989

ABSTRACT Isolated intact spinach chloroplasts were in- gradients as described by Douce and Joyard (10). We then cubated with phospholipase C (phosphatidylcholine choline- used the intact chloroplasts for envelope membrane and phosphohydrolase, EC 3.1.4.3) under mild experimental con- thylakoid purification and/or phospholipase C digestion ex- ditions in which only the phosphatidylcholine localized in the periments. cytosolic leaflet of the outer envelope membrane can be hy- Phospholipase C Treatment of Purified Intact Chloroplasts. drolyzed. Thylakoids, which were protected from phospholi- Phospholipase C (phosphatidylcholine cholinephosphohy- pase C degradation, were subsequently prepared from the drolase, EC 3.1.4.3.) from Bacillus cereus (grade 1, 4000 phospholipase C-treated chloroplasts and found to be devoid of units/ml) was purchased from Boehringer Mannheim. The phosphatidylcholine. Previously reported occurrences of phos- experiments were done essentially as described by Dorne et phatidylcholine in thylakoid preparations probably reflect al. (6). Intact and purified chloroplasts (final concentration, contamination ofthe thylakoids by envelope membranes. In the 1 mg of chlorophyll per ml) were incubated at 8°C for 3 min present work, contamination of thylakoids by envelope mem- in the following medium: 330 mM sorbitol/10 mM Tricine- branes was determined by measuring the 1,2-diacylglycerol NaOH, pH 7.8/0.3 unit of phospholipase C. Control exper- 3-fi-galactosyltransferase [monogalactosyldiacylglycerol iments were carried out under the same conditions. except (MGDG) synthase; UDPgalactose:1,2-diacylglycerol 3-.t-D-ga- that no phospholipase C was added to the mixture. The lactosyltransferase, EC 2.4.1.46] in the different digestion was terminated by the rapid cooling of the chloro- subfractions. We conclude that phosphatidylcholine is not plast suspension to almost 0°C; the incubation mixture was present in highly purified thylakoids. Phosphatidylcholine is layered on top of a 40% Percoll cushion containing 330 mM also absent from prokaryotic cyanobacterial membranes, and sorbitol and 10 mM Tricine-NaOH (pH 7.8). The tubes were our results are in agreement with the endosymbiotic origin of centrifuged as described by Joyard et al. (11) to remove both higher plant chloroplasts. the phospholipase C and the broken chloroplasts from the incubation mixture. We carefully verified that no phospho- lipase C remained in the pellet of intact chloroplasts. In According to the endosymbiotic theory, in eukary- addition, we carefully controlled the integrity of the chloro- otic cells could have originated as free-living organisms that plasts used for further analyses of thylakoid , and we found shelter within protoeukaryotes and then became sym- routinely used phase-contrast microscopy to monitor the biotic elements within them (1, 2). Comparison of the mem- integrity of the preparations. brane constituents in chloroplasts and cyanobacteria reveal Purification of Envelope Membranes and of Thylakoids. numerous similarities-i.e., the presence of galactolipids, Untreated and phospholipase C-treated chloroplasts were sulfolipid, and phosphatidylglycerol having the same struc- lysed in a hypotonic medium and the total envelope mem- ture-and a few striking differences, such as the absence of branes were purified from the lysate (swollen chloroplasts) by phosphatidylcholine, a typical eukaryotic , in cyanobac- centrifugation through a step gradient as described by Douce teria (3). Analyses of the lipid components in envelope et al. (7). The crude thylakoid fraction was recovered as a membranes from pea (4) and spinach (5) chloroplasts have pellet at the bottom of the tube. Washing of the thylakoids shown that large amounts of phosphatidylcholine (600-800 was done by resuspending the pellet with a solution contain- ,ug per mg ofprotein) are present only in the outer membrane, ing 330 mM sorbitol and 10 mM Tricine-NaOH (pH 7.8). The where it is concentrated in the cytosolic leaflet (6). In contrast, thylakoids were then recovered as a pellet by centrifugation all analyses ofthylakoids and ofthe inner envelope membrane for 5 min at 3000 x g. A series of washings were done to published so far indicate that phosphatidylcholine represents remove most of the contamination by chloroplast envelope from 3% to 10o of the total glycerolipids in these membrane membranes. fractions (4-9). It is assumed that this phospholipid is a Lipid Analyses. Total envelope and thylakoid lipids were genuine chloroplast component and its presence in internal extracted according to Bligh and Dyer (12). We determined membranes such as thylakoids has never been ques- the lipid composition by using thin-layer chromatography to tioned (for reviews, see refs. 8 and 9). The lack of phosphati- separate polar lipids and gas chromatography to analyze their dylcholine in cyanobacteria led us to probe whether phos- fatty acids (6). phatidylcholine was indeed a true component of thylakoid Assay of 1,2-Diacylglycerol 3-f8-galactosyltransferase. The membranes orjust a consequence of contamination by other activity of 1,2-diacylglycerol 3-,8-galactosyltransferase [mon- cell membranes containing phosphatidylcholine. ogalactosyldiacylglycerol (MGDG) synthase; UDPgalactose: 1,2-diacylglycerol 3-,3-D-galactosyltransferase, EC 2.4.1.46] MATERIALS AND METHODS was measured at 25°C by following the incorporation of galactose from UDP['4C]galactose into monogalactosyldia- Isolation of Purified Intact Chloroplasts. Chloroplasts were cylglycerol as described by Douce (13). The complete reac- isolated and purified by isopycnic centrifugation in Percoll tion mixture (200 ,ul) contained 20 mM Tricine-NaOH (pH

The publication costs of this article were defrayed in part by page charge Abbreviation: MGDG synthase, monogalactosyldiacylglycerol syn- payment. This article must therefore be hereby marked "advertisement" thase. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

71 Downloaded by guest on October 2, 2021 72 Botany: Dome et al. Proc. Natl. Acad. Sci. USA 87 (1990)

7.8), 1 mM MgCI2, and 0.5 mM UDP['4C]galactose (36.6 It is clear that washing of thylakoids led to parallel decreases Bq/nmol). After 10 min of incubation, 750 ,l of chloroform/ in the phosphatidylcholine content and in MGDG synthase methanol (1:2, vol/vol) was added to stop the reaction and activity in this membrane fraction. However, it was not extract the lipids. The labeled galactolipids were then ana- possible, using this procedure, to remove all the contaminat- lyzed according to Douce (13). ing envelope membranes. Therefore, the use of intact chlo- Assay of Photosynthetic Electron Flow in Thylakoids. The roplasts depleted of envelope phosphatidylcholine was nec- ability of thylakoids from phospholipase C-treated chloro- essary to determine the true level of phosphatidylcholine in plasts to catalyze photosynthetic reactions, using ferricya- thylakoids. This was achieved by using phospholipase C- nide and methyl viologen as electron acceptors, was moni- treated intact spinach chloroplasts (6). In agreement with our tored as described by Trebst (14) and Izawa (15). previous observations, Table 2 demonstrates that phospho- Chlorophyll and Determination. We measured chlo- lipase C digestion of intact chloroplasts led to a considerable rophyll concentrations in 80% acetone extracts (16) and decrease of the phosphatidylcholine level in chloroplast protein concentrations (17) with bovine serum albumin used envelope membranes. The decrease of the envelope phos- as a standard. phatidylcholine content was accompanied by an increase of the diacylglycerol content, thus demonstrating the enzymatic RESULTS AND DISCUSSION attack ofphosphatidylcholine by phospholipase C. As shown by Dome et al. (6), not all diacylglycerol formed during Preparation of Thylakoids Devoid of Envelope Phosphati- phospholipase C treatment of intact chloroplasts remain in dylcholine. It is possible to prepare chloroplasts devoid of the outer envelope membrane, but this does not affect the contaminating extraplastidial membranes from numerous membrane integrity (6). plant species (10, 18-20). Therefore, contamination by ex- Phosphatidylcholine Content of Thylakoids fromn Phospho- traplastidial phosphatidyicholine is rather easy to prevent. lipase C-Treated Intact Chloroplasts. The glycerolipid com- However, contamination of thylakoids by chloroplast enve- position of thylakoids from nontreated and from phospholi- lope membranes, containing phosphatidylcholine, is much pase C-treated spinach chloroplasts is given in Table 2. No more difficult to prevent (10). We have calculated (10) that difference between thylakoids from treated or nontreated about half of the envelope membranes (on a protein basis) chloroplasts was detected in their glycolipid content: galac- were recovered together with the thylakoid fraction when the tolipids and sulfolipid represented between 85% and 90%o of purified, intact chloroplasts were fractionated after an os- thylakoid glycerolipids. Nor was there any effect on phos- motic shock by sucrose gradient centrifugation according to phatidylglycerol content, the major phospholipid in thyla- Douce et al. (7). We have also demonstrated that both koids. In contrast, phosphatidylcholine represented -3% of envelope membranes fused together, which led to the for- thylakoid glycerolipids from nontreated chloroplasts, mation of a single population of mixed vesicles containing the whereas no phosphatidylcholine could be detected in thyla- two membranes (10). It is therefore highly likely that both koids from phospholipase C-treated chloroplasts. The disap- envelope membranes contaminate thylakoid preparations, pearance of phosphatidylcholine was not caused by hydrol- and not only the inner envelope membrane, as one would ysis of thylakoid phosphatidylcholine by phospholipase C, expect because of the higher density of the inner envelope since the chloroplast internal membranes were protected membrane compared to that ofthe outer membrane (10). It is from degradation by the outer envelope membrane (6). Sev- possible to remove most (but not all) of the contaminating eral lines of evidence demonstrate this fact. First, phospha- envelope membranes from thylakoids with a series of careful tidylglycerol, which is a good substrate for phospholipase C washings. For instance, Table 1 shows that MGDG synthase (6), was not destroyed in thylakoids (Table 2). Second, activity, a unique envelope marker enzyme (13), which can penetration of phospholipase C from B. cereus through the be detected in the crude thylakoid fraction, decreased to a two envelope membranes is highly unlikely because of its very low level in washed thylakoids. By using MGDG large mass (20 kDa). Third, Dome et al. (6) have demon- synthase activity in envelope membranes as a reference, the strated that the outer envelope membrane remained intact contamination ofwashed thylakoids by envelope membranes during the incubation and that the only chloroplast membrane could be estimated (on a protein basis) to =1% after one structure susceptible to phospholipase C degradation under washing. This figure is consistent with the lowest values of our experimental conditions was the cytosolic leaflet of the phosphatidylcholine content ofthylakoids after one washing. outer envelope membrane. Finally, we have verified that no Table 1. Galactolipid formation and phosphatidylcholine content in different thylakoid preparations (crude pellet and after washings) and in envelope membranes from spinach chloroplasts Thylakoids One Two Envelope Crude washing washings MGDG synthase nmol of galactose incorporated per hr per mg of protein 822 48 7.8 0.4 Phosphatidylcholine content ,ug of fatty acids per mg of protein 235 15 3 tr % total glycerolipids 20 2.9 0.6 tr Envelope membranes and a crude thylakoid fraction were prepared from purified intact spinach chloroplasts as described. Thylakoids were washed once or twice as described and the pellet was recovered for the MGDG synthase assay. The incorporation of galactose from UDPgalactose into galactolipids was assayed according to Douce (13). The phosphatidylcholine content was determined as described by Dome et al. (6) and is expressed as I.tg of fatty acids per mg of protein and % total glycerolipids in the fraction analyzed. The determination ofphosphatidylcholine was not really accurate for thylakoids after two washings because of its very low level (tr, traces-i.e., <0.5%). The analyses were reproduced at least three times. The values are from a representative experiment. Downloaded by guest on October 2, 2021 Botany: Dome et al. Proc. Natl. Acad. Sci. USA 87 (1990) 73 Table 2. Glycerolipid composition of thylakoids and envelope intact chloroplasts by phospholipase C had no physiological membranes before and after treatment of intact spinach effect on electron chain functioning in thylakoids. chloroplasts with phospholipase C from B. cereus In addition, our observation that all envelope phosphati- Envelope Thylakoids dylcholine was destroyed by phospholipase C treatment of intact chloroplasts suggests that the inner envelope mem- - PLC + PLC - PLC + PLC brane is also devoid of phosphatidylcholine. Unfortunately, Lipid ,ug % pug % lag % ,ug % separation of outer and inner envelope membranes (5) after MGDG 173 15 176 16 289 52 290 53 phospholipase C treatment of intact chloroplasts was not DGDG 370 32 363 33 144 26 148 27 possible (6) because of the high fragility of phospholipase TGDG 40 3.5 45 4 tr tr tr tr C-treated chloroplasts. Therefore, it was not possible to TTGDG 17 1.5 20 2 tr tr tr tr analyze pure inner envelope membrane. However, the lack of SL 80 7 75 7 39 7 38 7 phosphatidylcholine in the inner envelope membrane is also PtdCho 230 20 tr tr 16 3 0 0 supported by (i) the dramatic reduction of phosphatidylcho- PtdGro 86 7.5 75 7 39 7 38 7 line content of total envelope membranes and (ii) the imper- Ptdins 29 2.5 32 3 11 2 11 2 meability of the outer envelope membrane to phospholipase DG 126 11 286 26 tr tr tr tr C (6). This is additional evidence for an almost identical Total 1151 100 1086 99 554 100 548 100 glycerolipid composition of thylakoids and the inner enve- lope membrane (4, 5). Lipids (jug of fatty acids per mg of protein) were extracted and analyzed as described by Dome et al. (6). MGDG, monogalactosyl- These experiments suggest that, excepting the outer envelope diacylglycerol; DGDG, digalactosyldiacylglycerol; TGDG, trigalac- membrane, plastid membranes are devoid of phosphatidylcho- tosyldiacylglycerol; TTGDG, tetragalactosyldiacylglycerol; SL, sul- line. We do not know of another report of a eukaryotic mem- folipid; PtdCho, phosphatidylcholine; PtdGro, phosphatidylglycerol; brane that does not contain phosphatidylcholine. Therefore, our Ptdlns, phosphatidylinositol; DG, diacylglycerol; + or - PLC, with observations strongly support the analogy between chloroplasts or without phospholipase C treatment of isolated intact chloroplasts; and their prokaryotic counterparts, Prochloron (oxychlorobac- tr, traces (<1%). The analyses were reproduced at least three times. teria) and cyanobacteria. The lack of phosphatidylcholine in The values are from a representative experiment. thylakoids and in the inner envelope membrane from higher phospholipase C remained in the pellet of intact and purified plant chloroplasts is consistent with the absence of this phos- chloroplasts. For all these reasons, no hydrolysis ofthylakoid pholipid in prokaryotes that could be related to chloroplasts. phosphatidylcholine was possible after the incubation of The only difference in lipid composition between both is the intact chloroplasts in the presence of phospholipase C (6). strong enrichment of the cytosolic leaflet of the outer envelope Thus, we conclude that the absence ofphosphatidylcholine in membrane in phosphatidylcholine. Clearly, the physiological thylakoids was only due to the reduction of the level of function of phosphatidylcholine in the cytosolic leaflet of the contaminating phosphatidylcholine originating from the outer envelope membrane will prove to be very enlightening. outer envelope membrane. This conclusion is also supported We have demonstrated that it is possible to increase the amount by the presence in ihylakoids of trace amounts of diacylglyc- ofphosphatidylcholine within the outer envelope membrane by erol, trigalactosyldiacylglycerol, and tetragalactosyldiacyl- incubating intact chloroplasts with phosphatidyicholine lipo- glycerol. The formation of diacylglycerol and of these un- somes in the presence of purified phospholipid transfer protein natural galactolipids is due to the functioning, during the (23). Furthermore, these observations raise the question of the course of envelope preparation, of a galactolipid:galactolipid apparent lack oftransmembrane diffusion in the outer envelope galactosyltransferase, which is localized on the outer enve- membrane. Although it is a major component in the outer leaflet lope membrane (21, 22). The presence ofcontaminating outer of the outer envelope membrane, phosphatidylcholine is not envelope phosphatidylcholine in thylakoids is supported by redistributed to the thylakoids. The mechanism involved is the observation that fatty acids are similar in phosphatidyl- entirely unknown. choline from envelope membranes and from thylakoids (7). The conclusion that thylakoids are devoid of phosphatidyl- 1. Fredrick, J. F. (1981) Origins and Evolution of Eukaryotic Intracellular Organelles, Annals of the New York Academy of choline is also supported by the lack of difference in the Sciences (N.Y. Acad. Sci., New York), Vol. 361. biochemical and biological behavior of thylakoids with or 2. Schiff, J. A. (1982) On the Origins of Chloroplasts (Elsevier/ without phosphatidylcholine. For instance, the rate of pho- North-Holland, Amsterdam). tosynthetic electron flow was not affected in thylakoids from 3. Murata, N. (1987) in The Metabolism, Structure, and Function phospholipase C-treated intact chloroplasts. Using ferricya- ofPlant Lipids, eds. Stumpf, P. K., Mudd, J. B. & Nes, W. D. nide as an electron acceptor, the rate of uncoupled 02 (Plenum, New York), pp. 603-612. evolution was 260 ,umol of 02 per mg of chlorophyll per 4. Cline, K., Andrews, J., Mersey, B., Newcomb, E. H. & hr-i.e., identical to that measured in control thylakoids Keegstra, K. (1981) Proc. Natl. Acad. Sci. USA 78, 3595-3599. containing phosphatidylcholine. Furthermore, photophos- 5. Block, M. A., Dome, A.-J., Joyard, J. & Douce, R. (1983) J. phorylation by isolated thylakoids was in thylakoids Biol. Chem. 258, 13281-13286. identical 6. Dome, A.-J., Joyard, J., Block, M. A. & Douce, R. (1985) J. from nontreated or phospholipase C-treated chloroplasts. In Cell Biol. 100, 1690-1697. the absence of ADP, the oxygen consumption measured 7. Douce, R., Holtz, R. B. & Benson, A. A. (1973) J. Biol. Chem. during illumination of thylakoids in the presence of methyl 248, 7215-7222. viologen (which is due to the passive efflux of protons across 8. Harwood, J. L. (1980) in The Biochemistry of Plants, Lipids: the thylakoid membrane) was identical and low in both Structure and Function, ed. Stumpf, P. K. (Academic, New samples: rates of30-40 ,umol of 02 per mg of chlorophyll per York), Vol. 4, pp. 2-55. hr were obtained. After addition of ADP, the rate of oxygen 9. Mudd, J. B. (1980) in The Biochemistry of Plants, Lipids: consumption was increased to the same extent in both Structure and Function, ed. Stumpf, P. K. (Academic, New samples (50-60 ,umol of 02 per mg of chlorophyll per hr); York), Vol. 4, pp. 249-282. therefore, the ADP/0 ratio (='2) was identical in thylakoids 10. Douce, R. & Joyard, J. (1982) in Methods in Chloroplast Molecular Biology, eds. Edelman, M., Hallick, R. B. & Chua, from nontreated and phospholipase C-treated chloroplasts. N. H. (Elsevier/North-Holland, Amsterdam), pp. 239-256. These observations demonstrate (i) that the thylakoid prep- 11. Joyard, J., Billecocq, A., Bartlett, S. G., Block, M. A., Chua, arations used were highly intact, and (ii) that treatment of N. H. & Douce, R. (1983) J. Biol. Chem. 258, 10000-10006. Downloaded by guest on October 2, 2021 74 Botany: Dorne et al. Proc. Nadl. Acad. Sci. USA 87 (1990)

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