ROLE OF AN EXTRINSIC 33 KILODALTON PROTEIN OF PHOTOSYSTEM-II IN THE TURNOVER OF THE REACTION CENTER-BINDING PROTEIN D1 DURING PHOTOINHIBITION

The reaction center-binding protein D1 of photosystem II (PS II) under goes rapid turnover under light stress conditions. In the present stud y, we investigated the role of the extrinsic 33 kDa protein (OEC33) in the early stages of D1 turnover. D1 degradation was measured after st rong illumination (1000-5000 mu E m(-2) s(-1)) of spinach manganese-de pleted, PSII-enriched membrane and core samples in the presence and ab sence of the OEC33 under aerobic conditions at room temperature, PSII samples lacking the OEC33 were prepared by standard biochemical treatm ents with Tris or CaCl2/NH2OH while samples retaining the OEC33 were p repared with NH2OH or NaCl/NH2OH. The degradation of D1, monitored by SDS/urea-polyacrylamide gel-electrophoresis and Western blotting using specific antibodies against D1, proceeds to a greater extent in NH2OH -treated samples than in Tris-treated samples over a 60 min illuminati on period. Under the same conditions, significantly more aggregation o f D1 occurs in the Tris-treated samples than in the NH2OH-treated samp les. The lower level of D1 degradation in Tris-treated samples is not due to secondary proteolysis, as judged from the time course for degra dation at 25 degrees C or the degradation pattern at 4 degrees C, Simi larly, for NaCl/NH2OH-treated samples, D1 degradation is greater and D 1 aggregation less than in CaCl2/NH2OH treated samples. The effect of the presence of the OEC33 on D1 degradation and aggregation is confirm ed by reconstitution experiments in which the isolated OEC33 is restor ed back to Tris-treated samples, During very strong illumination, sign ificant loss of CP43 also occurs in Tris-treated but not in NH2OH-trea ted samples. Structural analysis of PS II core complexes by Fourier tr ansform infrared (FT-IR) spectroscopy revealed very little change in t he protein secondary structure after 10 min illumination of NH2OH-trea ted samples while a large 10% decrease of alpha-helix content occurs i n Tris-treated samples. On the basis of these results, we suggest that either (1) the OEC33 stabilizes the structural integrity of PS II suc h that it prevents the photodamaged D1 protein from aggregating with n earby polypeptides and thereby facilitating degradation or (2) the OEC 33 specifically stabilizes CP43, a putative D1-specific protease, whic h normally promotes the efficient degradation of D1.