Addition of C-terminal histidyl tags to PsaL and PsaKl proteins of cyanobacterial phtosystem I

Abstract

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<i>In vitro </i>mutagenesis was used to produce two photosystem I mutants of the cyanobacterium <i>Synechocystis </i>sp. PCC 6803. The mutants HK and HL contained hexahistidyl tags at the C-termini of the PsaKI and PsaL subunits, respectively.

The HK mutant contained wild-type amounts of trimeric PS I complexes, but the level of hexahistidine-tagged PsaK I 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 PsaKI 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.