Dionisi H M, Checa S K, Krapp A R, Arakaki A K, Ceccarelli E A, Carrillo N, Viale A M
Departamento de Microbiología, Universidad Nacional de Rosario, Argentina.
Eur J Biochem. 1998 Feb 1;251(3):724-8. doi: 10.1046/j.1432-1327.1998.2510724.x.
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.
DnaK系统对于在大肠杆菌中表达的豌豆叶绿体铁氧还蛋白-NADP⁺还原酶（FNR）的有效折叠是必需的。在表达还原酶的胞质前体（preFNR）或成熟脱辅基酶的大肠杆菌dnaK、dnaJ或grpE突变体中，成熟活性酶的形成严重受损，并且这些形式在这些细胞中大量聚集。在dnaK缺失突变体中，从多拷贝质粒共表达dnaK可恢复preFNR的加工和FNR的折叠，产生成熟大小的活性酶。GroESL伴侣蛋白的过表达未能阻止preFNR聚集，但它恢复了在表达成熟酶的dnaK缺失突变体中FNR的有效折叠。在缺乏OmpT蛋白酶的大肠杆菌细胞中表达preFNR导致未加工前体在细胞的可溶部分中积累。免疫沉淀研究证明了这种可溶的preFNR而非成熟还原酶与DnaK和GroEL的相互作用。我们得出结论，除了GroE伴侣蛋白[卡里略，N.，切卡雷利，E. A.，克拉普，A. R.，博吉奥，S.，费雷拉，R. G. & 维亚勒，A. M.（1992年）《生物化学杂志》267，15537 - 15541]外，DnaK伴侣系统在FNR的折叠途径中起关键作用。
