Iron regulatory factor expressed from recombinant baculovirus: conversion between the RNA-binding apoprotein and Fe-S cluster containing aconitase

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Abstract
Iron regulatory factor (IRF) Is a cytoplasmlc mRNA-blndlng protein that coordinates post-transcrlptlonally the expression of several important proteins in iron metabolism. Binding of IRF to Iron-responsive elements (IRE) in the 5′ untranslated region (UTR) of ferrltln and erythrold 5-aminolevullnic acid-synthase mRNAs inhibits their translation, whereas binding to IREs in the 3′ UTR of transfer-Tin receptor (TfR) mRNA prevents the degradation of this mRNA. IRF binds RNA strongly after Iron deprivation, but is Inactive, yet present, under conditions of high cellular iron supply. Recently, IRF was also shown to have aconltase activity indicating the existence of an Fe-S cluster In the protein. In the current study we expressed human IRF In Insect cells from recombinant baculovirus and analysed IRE-binding and aconltase activities under various culture conditions. Newly made apoprotein, synthesized in the absence of Iron, was fully active in IRE-bindlng, but showed no aconitase activity. In contrast, IRF made by cells grown in high iron medium bound RNA poorly, but exhibited high aconitase activity with a K m of 9.2 μ M for cls -aconltate. Apo-IRF was converted In vitro to active aconitase by Fe-S cluster-generating conditions, and under the same conditions lost Its RNA-blndlng capacity. These results indicate that the two activities are mutually exclusive and controlled through formation of the Fe-S cluster.