Intraplastidial lipid trafficking: Regulation of galactolipid release from isolated chloroplast envelope

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A method is described for cell-free studies of lipid release from isolated chloroplast envelope. The isolated membrane fraction incorporated radiolabeled galactose into galactolipids, predominantly monogalactosyldiacylglycerol, prior to immobilization of the membrane vesicles onto strips of nitrocellulose. The strips with immobilized membrane were individually incubated with various co-factors and the incubations were terminated by removing the strips. Radioactivity was determined for the strips with immobilized membrane as well as for the material released during the assay. The release of galactolipids from immobilized chloroplast envelope was time- and temperature dependent, required stroma protein(s) and was further stimulated by hydrolysable ATP, GTP and < 50 μM acyl-CoAs, of which 16:1-CoA was the most stimulative. To investigate whether guanine nucleotide-binding proteins could be involved, stroma and envelope were independently or together incubated with [α- 32 P]GTP or [γ- 32 P]GTP. Stroma and envelope proteins were phosphorylated and the envelope fraction contained GMP/GDP binding proteins as well. When the fractions were co-incubated, the patterns of protein phosphorylation and guanine nucleotide binding was different compared to the additive effects of the separate fractions, suggesting that guanine nucleotides may have roles in galactolipid release in addition to providing energy. The results point to several similarities between the regulation of galactolipid release from isolated chloroplast envelope and the regulation of vesicular trafficking among animal and yeast cytosolic membranes, although other mechanisms for lipid release cannot, at this stage, be ruled out.