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.
Tang
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Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
Chitnis
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PR
D001483:Base Sequence; D000458:Cyanobacteria / Q000737:chemistry*; D017931:DNA Primers; D006639:Histidine / Q000737:chemistry*; D016296:Mutagenesis; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000235:genetics; D045331:Photosystem I Protein Complex
D017931:DNA Primers; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; C082500:photosystem I, psaL subunit; D006639:Histidine