The PsaD subunit of photosystem I. Mutations in the basic domain reduce the level of PsaD in the membranes.
The PsaD subunit of photosystem I (PSI) is a peripheral protein that provides a docking site for ferredoxin and interacts with the PsaB, PsaC, and PsaL subunits of PSI. We used site-directed mutagenesis to determine the function of a basic region in PsaD of the cyanobacterium Synechocystis sp. PCC 6803. We generated five mutant strains in which one or more charged residues were altered. Western blotting showed that replacement of lysine (Lys)-74 with glutamine or glutamic acid led to a substantial decrease in the level of PsaD in the membranes. The mutant PSI complexes showed reduced NADP+ photoreduction activity mediated by ferredoxin; the decrease in activity correlated with the reduced level of PsaD. Using protein synthesis inhibitors we showed that the degradation rates of the mutant and wild-type PsaD were similar, indicating a defect in the assembly of the mutant protein. Treatment of the mutant PSI complexes with a different concentration of NaI showed that the mutations decreased affinity between PsaD and the transmembrane components of PSI. With glutaraldehyde, the mutant and wild-type PsaD proteins could be cross-linked with PsaC, but the PsaD-PsaL cross-linked product was reduced drastically when arginine-72, Lys-74, and Lys-76 were mutated simultaneously. These studies demonstrate that the basic residues in the central region of PsaD, especially Lys-74, are crucial in the assembly of PsaD into the PSI complex.
Chitnis
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Department of Biochemistry and Biophysics, Iowa State University, Ames 50011, USA. chitnis@iastate.edu
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017931:DNA Primers; D018698:Glutamic Acid; D005973:Glutamine; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000378:metabolism*; D045331:Photosystem I Protein Complex; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D046911:Macromolecular Substances; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D010940:Plant Proteins; C070290:PsaD protein, cyanobacteria; D005973:Glutamine; D018698:Glutamic Acid; D008239:Lysine