Localization of different photosystems in separate regions of chloroplast membranes

The stoichiometric amounts and the photoactivity kinetics of photosystem I (PSI) and of the alpha and beta components of photosystem II (PSII(alpha) and PSII(beta)) were compared in spinach chloroplast membrane (thylakoid) fractions derived from appressed and nonappressed regions. Stroma-exposed thylakoid fractions from the nonappressed regions were isolated by differential centrifugation following a mechanical press treatment of the chloroplasts. Thylakoid vesicles derived mainly from the appressed membranes of grana were isolated by the aqueous polymer two-phase partition method. Stroma-exposed thylakoids were found to have a chlorophyll a/chlorophyll b ratio of 6.0 and a PSII(beta)/PSI reaction center ratio of 0.3. Kinetic analysis of system II photoactivity revealed the absence of PSII(alpha) from stroma-exposed thylakoids. The photoactivity of system I in stroma-exposed thylakoids showed a single kinetic component identical to that of unfractionated chloroplasts, suggesting that PSI does not receive excitation energy from the PSII-chlorophyll ab light-harvesting complex. Thus, stroma-exposed thylakoids are significantly enriched in both PSI and PSII(beta). Inside-out vesicles from the appressed membranes of grana-partition regions had a chlorophyll a/chlorophyll b ratio of 2.0 and a PSII/PSI reaction center ratio of 10.0. The photoactivity of system II showed the membranes of the grana-partition regions to be significantly enriched in PSII(alpha). We conclude that PSII(alpha) is exclusively located in the membranes of the grana partitions while PSII(beta) and PSI are located in stroma-exposed thylakoids. The low PSI reaction center (P700) content of vesicles derived from grana partitions and the kinetic homogeneity of the PSI complex suggest total exclusion of P700 as a functional component in the membrane of the grana-partition region.