PHOSPHORYLATION OF THYLAKOIDS AND ISOLATED SUBTHYLAKOID VESICLES DERIVED FROM DIFFERENT STRUCTURAL DOMAINS OF THE THYLAKOID MEMBRANE FROM SPINACH CHLOROPLAST

(1)Thylakoids from spinach chloroplasts were phosphorylated, fragmented by sonication, and then fractionated by aqueous two-phase partitioning to yield membrane fragments, deriving from different structural domains of the membrane: grana, grana margins, grana core and stroma lamellae. The photochemical activities of PS I alpha and PS II alpha, located in the grana, and PS I beta and PS II beta, located in the stroma lamellae, were compared for phosphorylated and control thylakoids. The antenna size (reflected by the K-m value) and maximum activity (V-max) of PS II alpha declined by 19 and 23%, respectively, while for PS II beta the antenna size and V-max decreased by 4 and 12%, respectively. No significant changes in antenna size were detected for either grana PS I alpha or stroma lamellae PS I beta. Counter-current distribution was used for the quantitative separation of grana and stroma lamellae vesicles, Upon phosphorylation, the stroma lamellae fraction increased from 30% to 35% of the total, based on total absorbance at 680 nm. This increase can be explained by partial unstacking of the grana periphery and appressed membranes near the fret regions. Portions of the previously stacked membranes can therefore break and separate with the stroma exposed membrane. In addition, since the grana margins contain PS I alpha (with 40% larger antennae than PS I beta), which is functionally connected to LHC II, it is to be expected that some of these PS I alpha units will also enter the stroma lamellae fraction and thus help contribute to a lower chlorophyll a/b ratio and a small increase in the average PS I antenna size of the stroma lamellae fraction from phosphorylated thylakoids. It is concluded that the incidence of partial destacking of the grana, which occurs due to the phosphorylation of LHC II and PS II polypeptides, may promote the exposure of the granal PS I alpha centers to the aqueous stroma and increase cyclic electron flow around Photosystem I and thereby ATP production over NADPH production. (2) Subthylakoid vesicles, representing the different structural domains, were also examined for their properties following an incubation in presence of light and ATP. Phosphorylation of membrane proteins including LHC II and PS II associated polypeptides was observed in membrane fractions deriving from the grana lamellae and, to a lesser extent, the grana core. Three unidentified polypeptides of 15, 20 and 22 kDa were the most abundantly labeled polypeptides in the stroma lamellae fraction. No membrane proteins became phospho-labeled in the grana margin fraction.