Cooperation of the DnaK and GroE chaperone systems in the folding pathway of plant ferredoxin-NADP+ reductase expressed in Escherichia coli.
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.
Dionisi
H M
HM
Departamento de Microbiología, Universidad Nacional de Rosario, Argentina.
Checa
S K
SK
Krapp
A R
AR
Arakaki
A K
AK
Ceccarelli
E A
EA
Carrillo
N
N
Viale
A M
AM
D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018833:Chaperonins; D002736:Chloroplasts / Q000201:enzymology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins; D005287:Ferredoxin-NADP Reductase / Q000096:biosynthesis* / Q000737:chemistry*; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism*; D009876:Operon; D018532:Pisum sativum / Q000201:enzymology*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D001426:Bacterial Proteins; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D005287:Ferredoxin-NADP Reductase; D018833:Chaperonins; C063842:dnaK protein, E coli