Plant and Cell Physiology, 2001, Vol. 42, No. 12 1331-1337
© 2001 Oxford University Press
Identification of Functional Domains of the Extrinsic 12 kDa Protein in Red Algal PSII by Limited Proteolysis and Directed Mutagenesis
1 Department of Biology, Faculty of Science, Science University of Tokyo, Kagurazaka 1-3, Shinjuku-ku, Tokyo, 162-8601 Japan 2 Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, Yamazaki 2641, Noda, Chiba, 278-8510 Japan
The extrinsic 12 kDa protein in red algal photosystem II (PSII) functions to minimize the chloride and calcium requirement of oxygen-evolving activity [Enami et al. (1998) Biochemistry 37: 2787]. In order to identify functional domains of the 12 kDa protein, we prepared the 12 kDa protein lacking N-terminal peptides or C-terminal peptides or both by limited proteolysis and directed mutagenesis. The resulting 12 kDa protein fragments were examined for their binding and functional properties by reconstitution experiments. (1) A peptide fragment from Gly-6 to C-terminus of the 12 kDa protein was prepared by V8 protease. This fragment rebound to PSII completely, and it reactivated oxygen evolution partially in the absence of Cl– and Ca2+ ions but significantly in the presence of Cl– ion. (2) A peptide from Leu-10 to Phe-83 was obtained by chymotrypsin treatment. This peptide rebound to PSII effectively, but the rebinding did not restore oxygen evolution in both the absence and presence of Cl– and Ca2+ ions. (3) Two mutant proteins, one lacking five residues and the other lacking nine residues of the N-terminus, were able to bind to PSII effectively. Recovery of oxygen evolution by their binding was almost the same as that reconstituted with the V8 protease-treated peptide. (4) Three mutant proteins lacking ten, seven or three residues of the C-terminus effectively rebound to PSII, but their binding did not result in recovery of the oxygen evolution. In contrast, reconstitution with a mutant protein lacking one residue of the C-terminus showed the same high restoration of oxygen evolution as reconstitution with the full-length 12 kDa protein. (5) These results indicate that two residues from lysine of the C-terminus of the 12 kDa protein constitute an important domain for minimizing the chloride and calcium requirement of oxygen evolution. In addition, the N-terminus of the protein, at least five residues, has a secondary function for the chloride requirement.
3 Corresponding author: E-mail, enami@rs.noda.sut.ac.jp; Fax, +81-471-24-2150.
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