Do thylakoids really contain phosphatidylcholine?

Abstract

Isolated intact spinach chloroplasts were incubated with phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) under mild experimental conditions in which only the phosphatidylcholine localized in the cytosolic leaflet of the outer envelope membrane can be hydrolyzed. Thylakoids, which were protected from phospholipase C degradation, were subsequently prepared from the phospholipase C-treated chloroplasts and found to be devoid of phosphatidylcholine. Previously reported occurrences of phosphatidylcholine in thylakoid preparations probably reflect contamination of the thylakoids by envelope membranes. In the present work, contamination of thylakoids by envelope membranes was determined by measuring the 1,2-diacylglycerol 3-fi-galactosyltransferase [monogalactosyldiacylglycerol (MGDG) synthase; UDPgalactose:1,2-diacylglycerol 3-.t-D-galactosyltransferase, EC 2.4.1.46] in the different chloroplast subfractions. We conclude that phosphatidylcholine is not present in highly purified thylakoids. Phosphatidylcholine is also absent from prokaryotic cyanobacterial membranes, and our results are in agreement with the endosymbiotic origin of higher plant chloroplasts.According to the endosymbiotic theory, plastids in eukaryotic cells could have originated as free-living organisms that found shelter within protoeukaryotes and then became symbiotic elements within them (1, 2). Comparison of the membrane constituents in chloroplasts and cyanobacteria reveal numerous similarities-i.e., the presence of galactolipids, sulfolipid, and phosphatidylglycerol having the same structure-and a few striking differences, such as the absence of phosphatidylcholine, a typical eukaryotic lipid, in cyanobacteria (3). Analyses of the lipid components in envelope membranes from pea (4) and spinach (5) chloroplasts have shown that large amounts of phosphatidylcholine

Molecular aspects of plastid envelope biochemistry

“…For instance, phospholipid transfer proteins have been demonstrated to mediate net in vitro transfer of phosphatidylcholine between plant cell membranes, but it is not yet known whether such proteins are active in vivo (for a review see [24]). Finally, using isolated intact chloroplasts in which envelope phosphatidylcholine was removed by phospholipase C treatment, Dorne et al [16] demonstrated that thylakoids (and probably the inner envelope membrane) are also devoid of phosphatidylcholine, the presence of this phospholipid in thylakoid preparations probably being due to contaminating outer envelope membrane. These observations raise the problem of the apparent lack of transmembrane diffusion in the outer envelope membrane.…”

“…These observations raise the problem of the apparent lack of transmembrane diffusion in the outer envelope membrane. Although it is a major component in the outer leaflet of the outer membrane, phosphatidylcholine is not redistributed to the thylakoids [16]. The mechanism involved is entirely unknown.…”

“…In vitro, the galactolipid :galactolipid galactosyltransferase does not show strong specificity for any monogalactosyldiacylglycerol molecular species. However, if this enzyme is indeed the digalactosyldiacylglycerol-synthesizing enzyme, it should discriminate in vivo between the various monogalactosyldiacylglycerol molecular species that are available since (a) the proportion of eukaryotic molecular species is higher in digalactosyldiacylglycerol than in monogalactosyldiacylglycerol [18] and (b) digalactosyldiacylglycerol contains 16 : 0 fatty acids (up to 10-15%) at both the sn-1 and sn-2 position and very little 16 : 3 (in 16 : 3 plants), whereas monogalactosyldiacylglycerol contains little 16:0, but (in 16:3 plants) 16:3 at the sn-2 position [18].…”

“…Glycerolipid composition of plant cell membranes Except for analyses of prokaryotes indicated, this table summarizes data obtained in our laboratory on the glycerolipid composition of plant cell membranes [4,71. The presence of PtdCho in thylakoids and plastid inner envelope membrane is caused by contamination by the outer membrane [16]. Therefore the polar lipid composition of thylakoids and inner envelope membranes was recalculated to account for comtamination of these membranes by the outer envelope membrane.…”

Envelope Membranes from Spinach Chloroplasts Are a Site of Metabolism of Fatty Acid Hydroperoxides

“…A crude chloroplast pellet was obtained and purified further by isopycnic centrifugation in Perco11 gradients (Douce et al, 1973;Douce and Joyard, 1982). The Percoll-purified chloroplasts are devoid of phosphatidylethanolamine, indicating the absence of extraplastidial membranes Joyard, 1980, 1982;Dorne et al, 1990). Purified intact chloroplasts were then lysed in hypotonic medium, and the stroma, envelope membranes, and thylakoids were purified from the lysate by centrifugation through a step Suc gradient (Douce and Joyard, 1982).…”