Abstract
Lipid phosphorylation takes place within the chloroplast envelope. In addition to phosphatidic acid, phosphatidylinositol phosphate, and their corresponding lyso-derivatives, we found that two novel lipids underwent phosphorylation in envelopes, particularly in the presence of carrier-free [gamma-(32)P]ATP. These two lipids incorporated radioactive phosphate in chloroplasts in the presence of [gamma-(32)P]ATP or [(32)P]P(i) and light. Interestingly, these two lipids were preferentially phosphorylated in envelope membranes in the presence [gamma-(32)P]CTP, as the phosphoryl donor, or [gamma-(32)P]ATP, when supplemented with CDP and nucleoside diphosphate kinase II. The lipid kinase activity involved in this reaction was specifically inhibited in the presence of cytosine 5'-O-(thiotriphosphate) (CTPgammaS) and sensitive to CTP chase, thereby showing that both lipids are phosphorylated by an envelope CTP-dependent lipid kinase. The lipids were identified as phosphorylated galactolipids by using an acid hydrolysis procedure that generated galactose 6-phosphate. CTPgammaS did not affect the import of the small ribulose-bisphosphate carboxylase/oxygenase subunit into chloroplasts, the possible physiological role of this novel CTP-dependent galactolipid kinase activity in the chloroplast envelope is discussed.
Highlights
Lipid phosphorylation takes place within the chloroplast envelope
The lipid kinase activity involved in this reaction was inhibited in the presence of cytosine 5-O(thiotriphosphate) (CTP␥S) and sensitive to CTP chase, thereby showing that both lipids are phosphorylated by an envelope CTP-dependent lipid kinase
In a first report we have demonstrated the presence of four phosphorylated lipids in chloroplast envelope membranes, namely phosphatidic acid (PA), lysophosphatidic acid (LPA), PIP, and lysophosphatidylinositol phosphate (LPIP) [2]
Summary
CTP␥S did not affect the import of the small ribulose-bisphosphate carboxylase/oxygenase subunit into chloroplasts, the possible physiological role of this novel CTP-dependent galactolipid kinase activity in the chloroplast envelope is discussed. Cylglycerol; DGpDG, digalactosylphosphate diacylglycerol; LMGpDG, lysomonogalactosylphosphate diacylglycerol; MGDG, monogalactosyl diacylglycerol; MGpDG, monogalactosylphosphate diacylglycerol; NDPK, nucleoside diphosphate kinase; PEI, polyethyleneimine; PLA2, phospholipase A2; pSSu, precursor protein of the ribulose-bisphosphate carboxylase/ oxygenase small subunit; TLC, thin layer chromatography; HPLC, high pressure liquid chromatography; Mops, 4-morpholinepropanesulfonic acid; Tricine, N-tris(hydroxymethyl)methylglycine; ATP␥S, adenosine 5Ј-O-(thiotriphosphate); CTP␥S, cytosine 5Ј-O-(thiotriphosphate). The present report documents for the first time a CTP-dependent galactolipid kinase activity in chloroplast envelope membranes
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