Characterization of NarJ, a system-specific chaperone required for nitrate reductase biogenesis in Escherichia coli.
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.
Liu
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Department of Biochemistry and Molecular Biology, University of Texas Houston Medical School, Houston, Texas 77030, USA.
DeMoss
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D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000378:metabolism; D018832:Molecular Chaperones / Q000302:isolation & purification / Q000378:metabolism*; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D018832:Molecular Chaperones; D011994:Recombinant Proteins; D006639:Histidine; D009565:Nitrate Reductases; D050901:Nitrate Reductase