Lipid Phosphorylation in Chloroplast Envelopes

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 [ The lipid kinase activity involved in this reaction was specifically 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. The lipids were identified as phosphorylated galactolipids by using an acid hydrolysis procedure that generated galactose 6-phosphate. 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.One of the main functions assigned to the chloroplast is its ability to assimilate CO 2 under illumination. This key activity must be separated from the rest of the cell by a selective membrane barrier, namely the envelope, which is one of the three main plastid compartments in addition to thylakoids and the stroma. The envelope is constituted of two membranes, the inner and the outer envelopes, each having its own specific characteristic and property (1).Lipid phosphorylation occurs in chloroplast envelope membranes (2). The first identified lipids incorporating phosphate from [␥-32 P]ATP in isolated envelope vesicles were lysophosphatidic acid (LPA), 1 phosphatidic acid (PA), phosphatidylinositol phosphate (PIP), and lysophosphatidylinositol phosphate (LPIP). At the present time, the functions of these phospholipids are not known in chloroplasts, but: (i) they might produce substrates for lipid biosynthesis pathways; for instance, PIP can serve as substrate for a phosphatidylinositol-4,5-phosphate kinase and eventually can be hydrolyzed in second messengers like inositol 1,4,5-trisphosphate and DAG (3); (ii) they could directly interact with intracellular proteins to affect their location and/or activity; (iii) they might also change the local topology of other lipids, thereby modifying electrostatic interactions of membrane components. Indeed, in mammalian systems, LPA and PA, as minor cell lipids, are likely to act as potent activators of plasma membrane tyrosine kinase via G protein activation and intracellular protein kinases (4, 5). PA is the product of sn-diacylglycerol phosphorylation catalyzed by a diacylglycerol kinase (6). In plant signaling pathways, PA is also derived from PC via phospholipase D (7) and from diacylglycerol pyrophosphate (8). The production of LPA usually occurs after a first rapid accumulation of DAG, followed by phosphorylation, and activation of a PA-specific phospholipase A 2 within the plasma membrane (9). In plants, a diacylglycerol kinase from Arabidopsis thaliana (10) and LPA, a product of inducible PLA 2 (11), have been identified, but their specific role a...

Three Distinct Lipid Kinase Activities Are Present in Spinach Chloroplast Envelope Membranes: Phosphatidylinositol Phosphorylation Is Sensitive to Wortmannin and Not Dependent on Chloroplast ATP

“…It is noteworthy that in outer envelope membranes, the activity was much more sensitive to the drug (76% inhibition, compared to 43% inhibition in total envelope membranes, at a concentration of 0.5 M wortmannin). This observation is not surprising since PI and PI phosphorylated derivatives are known mainly confined to the outer envelope membranes (3,25). The third set of experiments consisted of separating the labeled PIP in total envelope membranes using a TLC system allowing the identification of PI 3 P from PI 4 P (23).…”

“…Indeed, chloroplasts, mitochondria, the endoplasmic reticulum, glyoxysomes and other vesicular membranes are all putative sites for lipid phosphorylation. Concerning the chloroplast, lipid phosphorylation occurs in envelope membranes, but not in thylakoid membranes, and galactolipids are substrates for CTP-dependent kinase(s) (2,3).…”

Human Dolichol Kinase, a Polytopic Endoplasmic Reticulum Membrane Protein with a Cytoplasmically Oriented CTP-binding Site

“…Microsomes were prepared, and DK activity was assayed for all cells as described under "Experimental Procedures." intermediates in mammals and plants (44,45) and the phosphorylation of galactolipids in chloroplast envelopes (46).…”

Transcription Factor Reb1p Regulates DGK1-encoded Diacylglycerol Kinase and Lipid Metabolism in Saccharomyces cerevisiae

“…The reaction mixture contained 50 m m Tris-HCl (pH 7.5), 0.1 m m dioleoyl-DAG, 1 m m Triton X-100, 1 m m CTP, 1 m m CaCl 2 , 10 m m 2-mercaptoethanol, and enzyme protein in a total volume of 0.1 ml. The [γ- 32 P]CTP used in the reaction was synthesized enzymatically from CDP and [γ- 32 P]ATP with nucleoside 5′-diphosphate kinase (60). β-Galactosidase activity was measured by following the formation of O- nitrophenyl from O- nitrophenyl β- d -galactopyranoside at A 410 nm (61).…”