Quality Control of Photosystem II
Quality Control of Photosystem II
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DOI：
10.1074/jbc.m602896200
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发表时间：
2006-08
影响因子：
4.8
通讯作者：
M. Yoshioka;Suguru Uchida;H. Mori;K. Komayama;S. Ohira;Noriko Morita;T. Nakanishi;Yasusi Yamamoto-Yasusi-Yama
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文献类型：
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作者：
M. Yoshioka;Suguru Uchida;H. Mori;K. Komayama;S. Ohira;Noriko Morita;T. Nakanishi;Yasusi Yamamoto-Yasusi-Yama
当菠菜类囊体受到中度热胁迫（40 °C，30分钟）时，氧气释放受到抑制，光系统II的反应中心结合蛋白D1发生裂解，产生23 kDa的N-末端片段。加入0.15 mm ZnCl 2和1 mm ATP可大大促进D1的切割，而1 mm EDTA可完全抑制D1的切割，表明ATP依赖性金属蛋白酶参与了D1的切割。除草剂3-（3，4-二氯苯基）-1，1-二甲基脲、溴苯腈和碘苯腈均与QB位点结合，抑制D1蛋白的切割，表明D1蛋白的DE环是热敏切割位点。我们通过用2 m KSCN处理类囊体来溶解蛋白酶，并通过明胶活性凝胶电泳在70-80-kDa区域中检测上清液中的蛋白酶活性。针对烟草FtsH和拟南芥FtsH 2的抗体与KSCN溶解级分的70-80-kDa条带反应，这表明级分中存在FtsH。根据这一发现，我们确定了同源拟南芥FtsH 8在70-80-kDa区域的基质辅助激光解吸电离飞行时间质量分析的类囊体。KSCN-溶解级分连续重建与类囊体显示热诱导切割的D1蛋白和生产的D1片段。这些结果有力地表明，FtsH蛋白酶参与了中度热应激下D1蛋白的初级切割。
When spinach thylakoids were subjected to moderate heat stress (40 °C for 30 min), oxygen evolution was inhibited, and cleavage of the reaction center-binding protein D1 of photosystem II took place, producing 23-kDa N-terminal fragments. The D1 cleavage was greatly facilitated by the addition of 0.15 mm ZnCl2 and 1 mm ATP and was completely inhibited by 1 mm EDTA, indicating the participation of an ATP-dependent metalloprotease(s) in the D1 cleavage. Herbicides 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, bromoxynil, and ioxynil, all of which bind to the QB site, inhibited the D1 cleavage, suggesting that the DE-loop of the D1 protein is the heat-sensitive cleavage site. We solubilized the protease by treating the thylakoids with 2 m KSCN and detected a protease activity in the supernatant by gelatin activity gel electrophoresis in the 70–80-kDa region. The antibodies against tobacco FtsH and Arabidopsis FtsH2 reacted with a 70–80-kDa band of the KSCN-solubilized fraction, which suggests the presence of FtsH in the fraction. In accordance with this finding, we identified the homolog to Arabidopsis FtsH8 in the 70–80-kDa region by matrix-assisted laser desorption ionization time-of-flight mass analysis of the thylakoids. The KSCN-solubilized fraction was successively reconstituted with thylakoids to show heat-induced cleavage of the D1 protein and production of the D1 fragment. These results strongly suggest that an FtsH protease(s) is involved in the primary cleavage of the D1 protein under moderate heat stress.