Cooperation of the DnaK and GroE chaperone systems in the folding pathway of plant ferredoxin-NADP+ reductase expressed in Escherichia coli.
Cooperation of the DnaK and GroE chaperone systems in the folding pathway of plant ferredoxin-NADP+ reductase expressed in Escherichia coli.
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DnaK 和 GroE 伴侣系统在大肠杆菌中表达的植物铁氧还蛋白-NADP 还原酶折叠途径中的合作。
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发表时间：
1998
期刊：
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通讯作者：
A. Viale
中科院分区：
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作者：
H. Dionisi;S. K. Checa;A. Krapp;A. Arakaki;E. Ceccarelli;N. Carrillo;A. Viale
DnaK系统是在大肠杆菌中表达的豌豆叶绿体铁氧还蛋白-NADP+还原酶（FNR）的生产性折叠所必需的。成熟活性酶的形成在E.大肠杆菌dnaK，dnaJ或grpE突变体表达的还原酶（前FNR）或成熟的脱辅基酶的胞质前体，这些形式广泛聚集在这些细胞。在dnaK无效突变体中，来自多拷贝质粒的dnaK的共表达恢复了前FNR的加工和FNR的折叠，从而形成成熟大小的活性酶。GroESL伴侣蛋白的过表达未能阻止preFNR聚集，但它恢复了表达成熟酶的dnaK无效突变体中FNR的生产性折叠。在OmpT蛋白酶缺陷的E.大肠杆菌细胞导致未加工的前体在细胞的可溶性部分中积累。免疫沉淀研究证明了这种可溶性preFNR，而不是成熟的还原酶，与DnaK和GroEL的相互作用。我们得出结论，除了GroE伴侣蛋白[Carrillo，N.，Ceccarelli，E.一、Krapp，A. R.，Boggio，S.，费雷拉河G. & Viale，A. M.（1992）J.Biol.Chem.267，15537-15541]，DnaK分子伴侣系统在FNR的折叠途径中起关键作用。
The DnaK system is required for the productive folding of pea chloroplast ferredoxin-NADP+ reductase (FNR) expressed in Escherichia coli. The formation of a mature active enzyme was severely impaired in E. coli dnaK, dnaJ or grpE mutants expressing either the cytosolic precursor of the reductase (preFNR) or the mature apoenzyme, and these forms aggregated extensively in these cells. Coexpression of dnaK from a multicopy plasmid in the dnaK-null mutants restored preFNR processing and folding of FNR, rendering a mature-sized active enzyme. Overexpression of GroESL chaperonins failed to prevent preFNR aggregation, but it restored productive folding of FNR in dnaK-null mutants expressing the mature enzyme. Expression of preFNR in OmpT-protease-deficient E. coli cells resulted in the accumulation of the unprocessed precursor in the soluble fraction of the cells. The interaction of this soluble preFNR, but not the mature reductase, with DnaK and GroEL was evidenced by immunoprecipitation studies. We conclude that, in addition to the GroE chaperonins [Carrillo, N., Ceccarelli, E. A., Krapp, A. R., Boggio, S., Ferreyra, R. G. & Viale, A. M. (1992) J. Biol. Chem. 267, 15537-15541], the DnaK chaperone system plays a crucial role in the folding pathway of FNR.