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 p rovides a docking site for ferredoxin and interacts with the PsaB, Psa C, and PsaL subunits of PSI. We used site-directed mutagenesis to dete rmine the function of a basic region in PsaD of the cyanobacterium Syn echocystis sp. PCC 6803. We generated five mutant strains in which one or more charged residues were altered. Western blotting showed that r eplacement of lysine (Lys)-74 with glutamine or glutamic acid led to a substantial decrease in the level of PsaD in the membranes. The mutan t PSI complexes showed reduced NADP(+) photoreduction activity mediate d by ferredoxin; the decrease in activity correlated with the reduced level of PsaD. Using protein synthesis inhibitors we showed that the d egradation rates of the mutant and wild-type PsaD were similar, indica ting 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 c omponents of PSI. With glutaraldehyde, the mutant and wild-type PsaD p roteins could be cross-linked with PsaC, but the PsaD-PsaL cross-linke d 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 crucia l in the assembly of PsaD into the PSI complex.