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PMID:9305880

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Citation

Liu, X and DeMoss, JA (1997) Characterization of NarJ, a system-specific chaperone required for nitrate reductase biogenesis in Escherichia coli. J. Biol. Chem. 272:24266-71

Abstract

The narGHJI operon encodes the three subunits, alpha, beta, and gamma, of the respiratory nitrate reductase complex in Escherichia coli. A fourth open reading frame of the operon encodes a putative protein, NarJ, which is not present in purified nitrate reductase, but is required for biogenesis of the membrane-bound complex. NarJ was identified with a T7 expression system and was produced at significantly less than stoichiometric levels relative to the three enzyme subunits. A functional His-tagged NarJ fusion protein was overexpressed from a multicopy plasmid, purified by Ni2+ affinity chromatography, and characterized. Western blot analysis with antibodies raised against the fusion protein demonstrated that NarJ remained in the cytosol after assembly of the active membrane complex. The cytosolic alphabeta complex accumulated in a narJ insertion mutant was rapidly degraded after induction, but was stabilized by NarJ expressed from a multicopy plasmid. Overproduction of the His-tagged NarJ fusion protein in the same mutant led to the formation of an alphabeta.NarJ complex, which was resolved by Ni2+ affinity chromatography. The NarJ protein therefore has the properties of a system-specific (private) chaperone that reacts directly with and modifies the properties of the cytosolic alphabeta subunit complex, but remains in the cytoplasm after the assembly of the active alphabetagamma complex in the membrane.

Links

PubMed

Keywords

Blotting, Western; Chromatography, Affinity; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Histidine/metabolism; Molecular Chaperones/isolation & purification; Molecular Chaperones/metabolism; Nitrate Reductase; Nitrate Reductases/biosynthesis; Nitrate Reductases/genetics; Nitrate Reductases/isolation & purification; Nitrate Reductases/metabolism; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ECOLI:NARJ

located_in

GO:0005737: cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

ECOLI:NARJ

part_of

GO:0005737: cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

ECOLI:NARJ

involved_in

GO:0051131: chaperone-mediated protein complex assembly

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

ECOLI:NARJ

involved_in

GO:0042128: nitrate assimilation

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete


See also

References

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