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 regions, were constructed in the cyanobacterium Synechocystis 6803 using site-directed mutagenesis. Integration and stability of the mutated D1 proteins in the thylakoid membrane were studied by immunoblot and pulse-chase analyses. It was found that in Δ(N325-E333), the D1 protein with a deletion in the C-terminal tail, could insert in the thylakoids to normal amounts but its stability in the membrane was dramatically reduced. Insertion of D1 in Δ(V58-D61) or Δ(D103-G109);G110R, with deletions in the A-B loop, was severely obstructed, For Δ(P350-T354), with a deletion in the processed region of the C-terminus of D1, no phenotypic effects were observed. The effects of failed D1 insertion or accumulation on Photosystem II assembly was monitored by immunoblot analysis. The conclusions from these experiments are that the extrinsic 33 kDa protein, CP43, and the β subunit of cytochrome b559 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.