Directed mutagenesis and structure-function analyses of the psbA gene products, D1 and D1', in the cyanobacterium Synechosystis 6803

Sammanfattning: Photosynthesis is the process by which the two essential requirements for man's survival on earth, food and oxygen, are produced. Oxygenic photosynthetic organisms, like plants, algae and the prokaryotic cyanobacteria, harness and convert the sunlight in specialized membranes called thylakoids. One of the protein complexes involved in the photosynthetic electron transport in the thylakoid membranes is Photosystem II (PSII). PSII is an oligomeric protein-pigment complex which consists of at least 20 polypeptide chains. Through a number of redox reactions, PSII couples the light-induced reduction of plastoquinone to the oxidation of H2O, thereby producing the O2 in the atmosphere. A key component of the PSII reaction center is the D1 protein. D1 is directly involved in most of the reactions related to the water splitting and charge separation during the primary photochemistry of PSII. Both synthesis and degradation of the D1 protein is stimulated by light, resulting in a very rapid turnover of the protein under high light conditions. Degradation of the D1 protein is a proteolytic event triggered by light-induced damage. Synthesis of the D1 protein occurs on thylakoid-bound ribosomes. After synthesis of the precursor D1 protein, a C-terminal extension is removed to form the mature, functional protein.The D1 protein is encoded by the psbA gene. In plants and algae the gene is located on the plastid genome and normally exists as a single copy while in cyanobacteria psbA belongs to a small multigene family. Being prokaryotes, cyanobacteria are excellent model systems for the study of oxygenic photosynthesis and the cyanobacterium Synechosystis 6803 has been widely used in this context. In Synechosystis 6803 there are three psbA genes, psbA1, psbA2 and psbA3. The psbA2 and psbA3 genes are homologous and produce an identical D1 protein while psbA1 is more divergent and is not transcribed.We are studying structure-function relationships of the D1 protein in Synechosystis 6803. Using site-directed mutagenesis, we have constructed some 50 mutants containing substitution as well as deletions. With some of the mutants we have shown that the translocation process of the D1 protein is very sensitive to perturbations of the amino acid sequence in the A-B loop. We have also demonstrated that a seven amino acid long C-terminal extension, specific for the precursor-D1 from cyanobacteria and red algae, does not affect processing of the D1 protein. Furthermore, we have shown that the assembly of the PSII complex depended on the translocation but not necessarily performance of the D1 protein. We have constructed mutations in the cytosol/stroma-exposed D-E loop of the D1 protein and presented results that point to the importance of this region in the proteolytic degradation of the D1 protein during photoinhibiton. We have also constructed several mutants with altered amino acids in the cytosol/stroma-exposed N-terminal and for the first time shown that the conserved amino acids in this head are not involved in the post-translational regulation of the D1 protein in Synechosystis 6803..Using site-directed mutagenesis, we managed to activate the silent psbA1 gene. The activation was performed by substituting the 160 nt long upstream psbA2 Bal1-Xba1 fragment for the 320 nt long upstream psbA1 Bal1-Xba1 fragment. Expression analyses showed that the 160 nt fragment of the psbA2 gene contains all information for the light activation and high light stimulation of psbA transcription in Synechosystis 6803 . The activated psbA1 gene, psbA1-K, produced a novel but functional D1 protein, D1'. D1' differs from the D1 protein in 56 of 360 amino acids.