SYNTHESIS AND ASSEMBLY OF THE D1 PROTEIN INTO PHOTOSYSTEM-II - PROCESSING OF THE C-TERMINUS AND IDENTIFICATION OF THE INITIAL ASSEMBLY PARTNERS AND COMPLEXES DURING PHOTOSYSTEM-II REPAIR

In previous studies [van Wijk, K. J,, Bingsmark, S,, Are, E,-M., & And ersson, B, (1995) J. Biol, Chem. 270, 25685-25695; van Wijk, K, J,, An dersson, B., & Are, E,-M, (1996) J, Biol. Chem 271, 9627-9636], we hav e demonstrated that D1 protein synthesized in isolated chloroplasts an d thylakoids is incorporated into the photosystem II (PSII) core compl ex. By pulse-chase experiments in these ia vitro systems, followed by sucrose gradient fractionation of solubilized thylakoid membranes, it was shown that this assembly proceeded stepwise; first the D1 protein was incorporated to form a PSII reaction center complex (PSII re), and through additional assembly steps the PSII core complex was formed. I n this study, we have analyzed this assembly process in more detail, w ith special emphasis on the initial events, through further purificati on and analysis of the assembly intermediates by nondenaturing Deripha t-PAGE and by flatbed isoelectric focusing, The D2 protein was found t o be the dominant PSII reaction center protein initially associating w ith the new D1 protein, This strongly suggests that the D2 protein is the primary ''receptor'' or stabilizing component during or directly a fter synthesis of the D1 protein. After formation of the D1-D2 heterod imer, cyt b(559) became attached, whereas the psbl gene product was as sembled as a subsequent step, thereby forming a PSII reaction center c omplex. Subsequent formation of the PSII core occurred by binding of C P47 and then CP43 to the PSII re. The rapid radiolabeling of a minor p opulation of a PSII core subcomplex without CP43 indicated that an ass ociation of newly synthesized D1 protein with a preexisting complex co nsisting of D2/cyt b(559)/psbl gene product/CP47 was possibly occurrin g, in parallel to the predominant sequential assembly pathway. The kin etics of synthesis and processing of the precursor D1 protein were fol lowed in isolated chloroplasts and were compared with its incorporatio n into PSII assembly intermediates. No precursor D1 protein was found in PSII core complexes, indicating either that incorporation into the PSII core complex facilitates the cleavage of the C-terminus or, more likely, that processing is more rapid than the assembly into the PSII core.