The involvement of cytosolic lipases in converting phosphatidyl choline to substrate for galactolipid synthesis in the chloroplast envelope

Here we report that cytosolic phospholipases are involved in the utilization of phosphatidylcholine (PC) as substrate for chloroplast-localized synthesis of monogalactosyldiacylglycerol (MGDG). Isolated chloroplasts were pre-incubated with lysoPC and 14C]18:0-CoA to form 14C]PC. When soluble plant proteins (cytosol) and UDP-galactose were added, 14C] MGDG was formed. An inhibitor of phospholipase D markedly lowered the formation of 14C]MGDG, whereas thermolysin pretreatment of the chloroplasts was without effect. The cytosolic activity resided in the >100-kDa fraction. In a second approach, 14C]PC-containing lipid mixtures were incubated with cytosol. Degradation of 14C]PC to 14C]diacylglycerol was highest when the lipid composition of the mixture mimicked that of the outer chloroplast envelope. We also investigated whether PC of extraplastidic origin could function as substrate for MGDG synthesis. Isolated chloroplasts were incubated with enriched endoplasmic reticulum containing radiolabelled acyl lipids. In the presence of cytosol and UDP-galactose, there was a time-dependent transfer of 14C]PC from this fraction to chloroplasts, where 14C]MGDG was formed. We conclude that chloroplasts recruit cytosolic phospholipase D and phosphatidic acid phosphatase to convert PC to diacylglycerol. Apparently, these lipases do not interact with chloroplast surface proteins, but rather with outer membrane lipids, either for association to the envelope or for substrate presentation.