Iron regulatory factor expressed from recombinant baculovirus: conversion between the RNA-binding apoprotein and Fe-S cluster containing aconitase.
Iron regulatory factor (IRF) is a cytoplasmic mRNA-binding protein that coordinates post-transcriptionally the expression of several important proteins in iron metabolism. Binding of IRF to iron-responsive elements (IRE) in the 5' untranslated region (UTR) of ferritin and erythroid 5-aminolevulinic acid-synthase mRNAs inhibits their translation, whereas binding to IREs in the 3' UTR of transferrin 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 aconitase 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 aconitase activities under various culture conditions. Newly made apoprotein, synthesized in the absence of iron, was fully active in IRE-binding, 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 Km of 9.2 microM for cis-aconitate. Apo-IRF was converted in vitro to active aconitase by Fe-S cluster-generating conditions, and under the same conditions lost its RNA-binding capacity. These results indicate that the two activities are mutually exclusive and controlled through formation of the Fe-S cluster.
Emery-Goodman
A
A
Swiss Institute for Experimental Cancer Research, Genetics Unit, Epalinges.
Hirling
H
H
Scarpellino
L
L
Henderson
B
B
Kühn
L C
LC
D000154:Aconitate Hydratase / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001059:Apoproteins / Q000378:metabolism; D016367:Baculoviridae; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D066298:In Vitro Techniques; D007313:Insecta; D007501:Iron / Q000378:metabolism*; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D001059:Apoproteins; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D007501:Iron; D000154:Aconitate Hydratase