The PsaD Subunit of Photosystem I ' Mutations in the Basic Domain Reduce the Leve 1 of PsaD in the Membranes

Fd. PsaD and PsaE facilitate Fd docking, and PsaE may be The PsaD subunit of photosystem I (PSI) i s a peripheral protein involved in cyclic electron flow "nd PSI (Chimis, 1996). that provides a docking site for ferredoxin and interacts with the PsaD is a crucial component on the reducing side of PSI. PsaB, PsaC, and PsaL subunits of PSI. We used site-directed muThe insertional inactivation of the psaD gene in the PSI tagenesis to determine the function of a basic region in PsaD of the complexes of the cyanobacterium Syneckocystis sp. PCC cyanobacterium Synechocystis sp. PCC 6803. We generated five 6803 indicated that PsaD is essential for efficient function of mutant strains in which one or more charged residues were altered. the cyanobacterial p s ~ (Chimis et al., 1989). PsaD has sevWestern blotting showed that replacement of IYSine (LYS)-74 with era1 roles in the function and organization of PSI. First, it glutamine or glutamic acid led to a substantial decrease in the leve1 interacts with at least three proteins of PSI and stabilizes of PsaD in the membranes. The mutant PSI complexes showed their organization within the complex. cross-1hking experreduced NADP+ photoreduction activity mediated by ferredoxin; iments have demonstrated a close association of PsaD with PsaC and PsaL (Xu et al., 1994a; Armbrust et al., 1996; the decrease in activity correlated with the reduced leve1 of PsaD. Using protein synthesis inhibitors we showed that the degradation Jansson et al., 1996). Limited proteolysis experiments rates of the mutant and wild-type PsaD were similar, indicating a defect in the assembly of the mutant protein. Treatment of the showed that lo0Ps Of mutant PSI complexes with a different concentration of Na1 showed (sun et 1997b). Secondj influentes assembly Of that the mutations decreased affinity between PsaD and the transPsaC into paramagnetic membrane components of PSI. With glutaraldehyde, the mutant and reSOnance ProPerties of the terminal electron donors FA wild-type PsaD proteins could be cross-linked with PsaC, but the and FB (Chitnis et al., 1996; Hanley et a]., 1996). T h i d PsaD-PsaL cross-linked product was reduced drastically when PsaD is an essential component of the docking site for Fd. arginine-72, Lys-74, and Lys-76 were mutated simultaneously. It can be cross-linked to Fd using a hydrophilic, zeroThese studies demonstrate that the basic residues in the central length cross-linker, N-ethyl-3(3-dimethylaminopropyl)region of PsaD, especially Lys-74, are crucial in the assembly of carbodiimide (Zanetti and Merati, 1987; Zilber and Malkin, PsaD into the PSI complex. 1988); the cross-linked product is redox-active (Lelong et al., 1996). The Lys-106 residue of PsaD in Synechocystis sp. PCC 6803 can be cross-linked with the Glu-93 residue of Fd (Lelong et al., 1994). The PsaD-less mutants of Syneckocystis sp. PCC 6803 were used to demonstrate the functional significance of PsaD in electron transfer to Fd (Xu et al., 1994c; Hanley et al., 1996). The Lys-106 residue is a dispensable component of the docking site, and an ionic interaction between Lys-106 of PsaD and Glu-93 of Fd is not essential for electron transfer to Fd (Chitnis et al., 1996; Hanley et al., 1996). Analysis of site-directed mutations in