Abstract

The galactolipid transfer from inner envelope to thylakoid membranes has been studied in intact spinach chloroplasts. Plastids, isolated from mature leaves, were dark-incubated in the presence of UDP-[14C]galactose. After various synthesis periods at 5 or 25 degrees C, intact plastids were reisolated and osmotically lysed. Thylakoid membranes were then prepared by a special procedure which removed greater than or equal to 99% of the envelope amount initially present. Under these conditions, purified thylakoids were found to contain radiolabelled MGDG and DGDG, indicating that galactolipids were exported from the inner envelope. The amounts exported were proportional to the amounts synthesized. About 55% of the MGDG and 25% of the DGDG synthesized in plastids were transferred to thylakoids, irrespectively of incubation time or temperature. The MGDG/DGDG radioactivity ratio was 7 in intact plastids and 18 in thylakoids, suggesting a preferential export of MGDG. Purified thylakoid membranes were then submitted to a lipolytic treatment designed to discriminate between the MGDG and DGDG pools belonging to the outer (stroma-facing) or to the inner monolayer. The radioactivity present in the lyso-products (corresponding to the outer pools) and in the residual parent lipids (corresponding to the inner pools) was measured. The labelled MGDG showed a transmembrane outside:inside distribution (mol%) of 50:50, which differed from the native (mass) MGDG asymmetry of 64:36. In contrast, the label and mass asymmetries of DGDG gave the same value of 15:85. These label distributions were affected neither by incubation time (from 5 to 90 min) nor by temperature (from 5 to 25 degrees C). We discuss the possibilities that transient fusions between the stroma-facing monolayers of the inner envelope and of the thylakoid membrane, and/or galactolipid transfer protein(s), together with lipid translocating activities in thylakoids, may account for the galactolipid export observed in mature spinach chloroplasts.