The carboxyl-terminal extension of the precursor D1 protein of photosystem II is required for optimal photosynthetic performance of the cyanobacterium Synechocystis sp. PCC 6803

Abstract

The D1 protein is an integral component of the photosystem II reaction center complex. In the cyanobacterium Synechocystis sp. PCC 6803, D1 is synthesized with a short 16-amino acids-long carboxyl-terminal extension. Removal of this extension is necessary to form active oxygen-evolving photosystem II centers. Our earlier studies have shown that this extension is cleaved by CtpA, a specific carboxyl-terminal processing protease. The amino acid sequence of the carboxyl-terminal extension is conserved among D1 proteins from different organisms, although at a level lower than that of the mature protein. In the present study we have analyzed a mutant strain of Synechocystis sp. PCC 6803 with a duplicated extension, and a second mutant that lacks the extension, to investigate the effects of these alterations on the function of the D1 protein in vivo. No significant difference in the growth rates, photosynthetic pigment composition, fluorescence induction, and oxygen evolution rates was observed between the mutants and the control strain. However, using long-term mixed culture growth analysis, we detected significant decreases in the fitness of these mutant strains. The presented data demonstrate that the carboxyl-terminal extension of the precursor D1 protein is required for optimal photosynthetic performance.

Figure 1

The general scheme for the construction of the mutants. Various restriction sites are indicated. A, Partial nucleotide sequence of the psbA2 gene in the wild-type strain, which corresponds to the carboxyl-terminal extension of the D1 protein. The oligonucleotide primers used for PCR amplification are shown by small horizontal arrows. B, Construction of plasmids for the generation of various mutant strains. See text for further details.

Figure 2

Immunoblot analysis of membrane proteins from cells of the control strain and mutants. The blot was probed with antibodies raised against the D1 protein. Positions of the mature D1 protein and its precursor form (pD1) are shown on the right.

Figure 3

Schematic diagram of the psbA2 gene and its downstream region in constructed mutants in comparison with that from the control strain. The oligonucleotide primers used for PCR amplification in long-term mixed-culture experiments are shown as horizontal arrows. Sizes of PCR products are shown on the right side. A, WT^K and MatD1 strains; B, WT^K and DoubleExt strains.

Figure 4

Patterns of amplified PCR products, which were separated by agarose gel electrophoresis, from the mixed-culture cells. Names of the strains and size of PCR products are marked on the left side of the corresponding bands. The conditions under which the mixed cultures were grown are indicated on the right side. The numbers 0 to 6 refer to the number of times of subculturing. A, WT^K and MatD1 mixed-culture. B, WT^K and DoubleExt mixed-culture. C, WT^K and DoubleExt mixed culture in the presence of DCMU and Glc.

Figure 5

The relative amounts of the PCR products in the mixed-culture experiments. Each experiment was repeated three times and standard deviations are indicated. A, Relative amounts of PCR products for WT^K (solid line) and MatD1 (dashed line) mutants in the mixed culture experiment under high light. B, Relative amounts of PCR products from WT^K (solid line) and DoubleExt (dashed line) mutants in the mixed culture experiment under dim light in the presence of DCMU and Glc. See text and Figure 4 for further details.