Processing of Precursor D1 Protein in the PSII Membrane by CtpA Over-Expressed in E. coli — An Analysis Using an Antibody Which Specifically Recognizes the Cleavage Product
In eukaryotic organisms, the D1 protein (D1) of the photosystem II (PSII) reaction center (RC) is encoded by the chloroplast DNA. The D1 is synthesized at the stromal side of the thylakoid membrane as a precursor form (pD1) with a C-terminal extension consisting of 8-16 amino acids which is co-translationally translocated into the lumenal space. The post-translational cleavage of the extension occurs in the lumenal space by a nuclear-encoded endo-peptidase imported from the cytosol. This process is absolutely essential to the integration of the Mn-cluster of the PSII, as shown by earlier studies on the LF-1 mutant of Scenedesmus  and site-directed mutants of Synechocystis . A novel serine-type protease involved in the processing has been identified, and a nuclear gene coding for the enzyme (ctpA gene) has been sequenced [3–8]. The predicted amino acid sequence revealed that the protease possesses a bipartite pre-sequence, consistent with the biochemical analysis for its location in chloroplasts. In previous studies, we utilized two types of substrate for the analysis of enzyme-substrate interaction, i.e., (i) the synthetic oligopeptide corresponding to the C-terminal sequence of pD1 , and (ii) the in vitro translated full-length pD1 . An unexpected finding from these studies was that the proteolytic activity exhibits a drastic pH dependency with an optimum in the pH region of 7.5–8.0, and this activity is quite low on the acidic side, i.e., 5.0–6.0; the pH range of lumenal space in illuminated thylakoids in which the activity for the C-terminal processing protease is expected to be extremely high. The present study was intended to analyze this inconsistency in pH dependency. For this purpose, we utilized pD1 in the PSII complex in the thylakoid membrane from the LF-1 mutant of Scenedesmus obliquus in which the processing protease is inactive. For purpose of analysis, we have developed an over-expression system for the protease in order to obtain substantial amounts of CtpA in its active state, and a preparation of anti-D1 antibody which specifically recognizes the C-terminus of mature D1 (mD1) produced by the action of processing protease.
Key wordsbiosynthesis pH dependence processing enzyme protein assembly reaction center thylakoid membranes
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