No abstract

Three spinach chloroplast envelope membrane preparations (i.e. whole, outer and inner membranes) were incubated in the presence of [γ-32 Ρ]ΑΤΡ. After lipid extraction and separation by TLC, four main phosphorylated lipids were detected by autoradiography in whole envelope preparations. These phospholipids were identified by comparing their Rf with that of lipid markers and by a deacylation procedure. They were found to be phosphatidic acid (PA) and lyso-PA, L-a-phosphatidyl-inositol 4-monophosphate (PIP) and lyso-PIP. These lipids were not equally distributed in the outer and inner envelope membranes. Chloroplast envelope membranes were verified not to be contaminated by plasma membranes. It is concluded that lipid kinase activities are associated with spinach chloroplast envelope membranes.© 1997 Federation of European Biochemical Societies.

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...

Abstract. Two chloroplast envelope proteins from spinach (Spinacia oleracea L.) exhibiting relative molecular masses (Mrs) of 26 and 14 kDa are apparently phosphorylated by a unique Ca2+-dependent serine protein kinase. The activity of this enzyme shows the same sensitivity towards pH, Ca 2+, Mg 2+, H7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine] and ATP concentrations (Siegenthaler and Bovet 1993, Planta 190, 231-240). Autoradiographic analyses following two-dimensional-gel electrophoresis (isoelectric focusing and SDS-PAGE) associated with Western blotting experiments indicate that these two phosphoproteins appeared to be pools of the light-harvesting complex of photosystem II (LHCII) and of the ribulose-l,5-bisphosphate carboxylase-oxygenase (Rubisco) small subunit, respectively. Immunoprecipitation of envelope-phosphorylated proteins, using immunoglobulins (IgG) directed to the apoprotein of LHCII and to the holoenzyme of Rubisco confirmed that LHCII and the Rubisco small subunit effectively incorporated 3ap from (7-32p)ATP in isolated envelope membranes. We propose that, in agreement with the fact that protein import is driven by ATP, the phosphorylation of LHCII and the Rubisco small subunit could take place after the processing of precursor proteins and could be an obligatory step for their internalization into chloroplasts. Key words: