Do Thylakoids Really Contain Phosphatidylcholine?
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Proc. Nati. Acad. Sci. USA Vol. 87, pp. 71-74, January 1990 Botany Do thylakoids really contain phosphatidylcholine? (chloroplasts/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 Grenoble 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 chloroplast 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, plastids 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 lipids, 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 lipid, 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 plastid 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 Protein 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