No abstract

The D1 protein of the photosystem II (PSII) complex in the thylakoid membrane ofoxygenic photosynthetic organisms is synthesized as a precursor polypeptide (pDl) with a C-terminal extension. Posttransational processing ofthe pDl protein is essential to estbfsh water oxidation activity of the PSII complex. We have recently identified a gene, cipA, a mutation in which resulted in a loss of PSII activity in the cyanobacterium Synechocystis sp. PCC 6803. To study the function of the CtpA protein, we inactivated the ctpA gene by inserting a kanamycin-resistance gene into its coding sequence. The resultant mutant strain, T564, had no PSII-mediated water oxidation activity, but it had normal cytochrome b6fand photosystem I activities. Measurements ofthermoluminescence profiles and rates of reduction of 2,6-dichilorophenolindophenol indicated that PSI1 complexes in the mutant cells had functional reaction centers that were unable to accept electrons from water. Immunoblot analysis showed that D1, D2, CP47, CP43, and the a subunit of cytochrome bwg, five Integral membrane proteins of PSI1, were present in T564 cells. Interestingly, the D1 protein in the mutant cells was 2 kDa larger than that in wild-type cells, due to the presence of a C-terminal extension. We conclude that the CtpA protein is a processing enzyme that cleaves off the C-terminal extension of the D1 protein. Interestingly, the CtpA protein shows scant sequence similarity to the interphotoreceptor retinoid-binding proteins in the bovine, human, and insect eye systems.

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins.