PLANT PHYSIOLOGY , Vol 115, Issue 4 1699-1705, Copyright © 1997 by American Society of Plant Biologists
The PsaD Subunit of Photosystem I (Mutations in the Basic Domain Reduce the Level of PsaD in the Membranes)
V. P. Chitnis, A. Ke and P. R. Chitnis
Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011
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 Nal 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.
