Characterization of NarJ, a system-specific chaperone required for nitrate reductase biogenesis in Escherichia coli

Abstract

The narGHJI operon encodes the three subunits, α, β, and γ, 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 αβ 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 αβ-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 αβ subunit complex, but remains in the cytoplasm after the assembly of the active αβγ complex in the membrane.