Chloroplast import of four carotenoid biosynthetic enzymes in vitro reveals differential fates prior to membrane binding and oligomeric assembly
Chloroplast import of four carotenoid biosynthetic enzymes in vitro reveals differential fates prior to membrane binding and oligomeric assembly
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DOI：
10.1111/j.1432-1033.1997.00942.x
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发表时间：
1997-08-01
期刊：
影响因子：
--
通讯作者：
Beyer, P
中科院分区：
文献类型：
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作者：
Bonk, M;Hoffmann, B;Beyer, P
将类胡萝卜素生成酶香叶基香叶基二磷酸合酶、八氢番茄红素合酶、八氢番茄红素去饱和酶和番茄红素环合酶的前体蛋白导入豌豆叶绿体。牛儿基牛儿基二磷酸合酶保持可溶于基质中的游离形式和八氢番茄红素合酶相关的类囊体膜进口后，两者都如预期。令人惊讶的是，八氢番茄红素去饱和酶和番茄红素环化酶，强烈依赖于膜协会的酶活性，也保持可溶于叶绿体基质。然而，这些酶的可溶性形式仍然能够进行膜结合。例如用不含蛋白质的脂质体膜。实际上，八氢番茄红素合酶、八氢番茄红素去饱和酶和番茄红素环化酶的可溶形式作为ATP和冷敏感性高分子量复合物出现。凝胶过滤实验和蓝色非变性PAGE加放射自显影和蛋白质印迹分析表明叶绿体60-kDa伴侣蛋白（Cpn 60）参与了输入的胡萝卜素生成酶的可溶性高分子量复合物。最后，据推测，膜结合调节因子在膜结合中起决定性作用。
The precursor proteins of the carotenogenic enzymes geranylgeranyl diphosphate synthase, phytoene synthase, phytoene desaturase and lycopene cyclose were imported into isolated pea chloroplasts. Geranylgeranyl diphosphate synthase remained soluble in the stroma in a free form and phytoene synthase associated to thylakoid membranes upon import, both as expected. Surprisingly, phytoene desaturase and lycopene cyclase, which strongly depend on membrane association for enzymatic activity, also remained soluble in the chloroplast stroma. The soluble forms of these enzymes were, however, still competent for membrane-association. e.g. with protein-free liposomal membranes, Indeed the soluble forms of phytoene synthase, phytoene desaturase and lycopene cyclase occurred as ATP- and cold-sensitive high-molecular-mass complexes, Gel-filtration experiments and blue native-PAGE plus autoradiography and western blot analysis indicated a participation of the chloroplast 60-kDa chaperonin (Cpn60) in the soluble high-molecular-mass complexes of imported carotenogenic enzymes. Finally, it was inferred that a membrane-bound regulatory factor plays a decisive role in membrane-binding.