CONSTRUCTED DELETIONS IN LUMEN-EXPOSED REGIONS OF THE D1 PROTEIN IN THE CYANOBACTERIUM SYNECHOCYSTIS-6803 - EFFECTS ON D1 INSERTION AND ACCUMULATION IN THE THYLAKOID MEMBRANE, AND ON PHOTOSYSTEM-II ASSEMBLY

Modified forms of the D1 protein with deletions in lumen-exposed regio ns, were constructed in the cyanobacterium Synechocystis 6803 using si te-directed mutagenesis. Integration and stability of the mutated D1 p roteins in the thylakoid membrane were studied by immunoblot and pulse -chase analyses. It was found that in Delta(N325-E333), the D1 protein with a deletion in the C-terminal tail, could insert in the thylakoid s to normal amounts but its stability in the membrane was dramatically reduced. Insertion of D1 in Delta(V58-D61) or Delta(D103-G109);G110R, with deletions in the A-B loop, was severely obstructed, For Delta(P3 50-T354), with a deletion in the processed region of the C-terminus of D1, no phenotypic effects were observed. The effects of failed D1 ins ertion or accumulation on Photosystem II assembly was monitored by imm unoblot analysis. The conclusions from these experiments are that the extrinsic 33 kDa protein, CP43, and the beta subunit of cytochrome b55 9 accumulate in the thylakoid membrane independently of the D1 protein , and that accumulation of the D2 protein and CP47 requires insertion but not necessarily accumulation of the D1 protein.