Addition of C-terminal histidyl tags to PsaL and PsaK1 proteins of cyanobacterial photosystem I.

In vitro mutagenesis was used to produce two photosystem I mutants of the cyanobacterium Synechocystis sp. PCC 6803. The mutant HK and HL contained hexahistidyl tags at the C-termini of the PsaK1 and PsaL subunits, respectively. The HK mutant contained wild-type amounts of trimeric PS I complexes, but the level of hexahistidine-tagged PsaK1 was found only ten per cent in the PS I complexes and membranes of the wild type level. Therefore, attachment of a tag at the C-terminus interferes with the expression or assembly of PsaK1. In contrast, the HL mutant contained a similar level of tagged PsaL as that in the wild type. However, trimeric PS I complexes could not be obtained from this strain, indicating that the C-terminus of PsaL is involved in the formation of PS I trimers. Hexahistidine-tagged complexes of the HL and HK strains could not be purified with Nickel-affinity chromatography, unless photosystem I was denatured with urea, demonstrating that tagged C-termini of PsaK1 and PsaL were embedded inside of the PS I complex. Protection of the C-terminus from trypsin cleavage further supported this conclusion. Thus, histidine tagging allowed us to demonstrate role of C-termini of two proteins of photosystem I.

Proteomics: a powerful tool in the post-genomic era.

Genomics is having a profound impact on biological research, including photosynthesis investigations. Genomes of many photosynthetic organisms have been sequenced. The information about ALL genes that govern and execute photoautotrophic metabolism provides many opportunities to understand genome function and details of known and uncharted pathways. Proteomics, analysis of the protein complement of the genome, is a powerful tool in understanding which proteins are present in a particular tissue under given conditions. Proteomics also allows us to estimate relative levels of proteins and to determine post-translational modifications of the gene products. In this minireview, we discuss the technology and its applications in plant sciences.