Abstract
The galactolipids monogalactosyldiglyceride and digalactosyldiglyceride together comprise more than 77% of the photosynthetic membrane lipids of higher plant chloroplasts. We have isolated a lipase from the chloroplasts of runner beans (Phaseolus vulgaris) which is highly specific for these galactolipids. This galactolipase promotes the hydrolysis of monogalactosyldiglyceride and digalactosyldiglyceride, in the process liberating two free fatty acids into the membrane bilayer, leaving the residual galactosyl glyceride group to diffuse into the aqueous bulk phase. Isolated spinach photosynthetic membranes were treated with this enzyme preparation and changes in membrane composition were studied with thin layer chromatography (for lipids), gel electrophoresis (proteins), and freeze-etching (membrane structure). After 30 min of lipolysis, nearly 100% of the galactolipids had been converted into membrane-associated fatty acids and water-soluble galactosyl glycerides. SDS PAGE showed that two proteins, one of which is possibly associated with the reaction center of photosystem II, were removed by the treatment. Despite the minor nature of changes in membrane protein composition, freeze-fracture and freeze-etch studies showed that striking changes in membrane structure had taken place. The large freeze-fracture particle on the E fracture face had disappeared in stacked regions of the membrane system. In addition, a tetrameric particle visible at the inner surface of the membrane had apparently dissociated into individual monomeric particles. The fact that these two structures are so dramatically affected by the loss of galactolipids strongly suggests that these lipids play a crucial role in maintaining their structure. Both structures are believed to be different views of the same transmembrane unit: a membrane-spanning complex associated with photosystem II. Our results are consistent with two possible interpretations: the intramembrane particles may be lipidic in nature, and hence lipolysis causes their disappearance; or galactolipids are necessary for the organization of a complex photosystem II-associated structure which is composed of a number of different molecular species.