Mutational analysis of the [4Fe-4S]-cluster converting iron regulatory factor from its RNA-binding form to cytoplasmic aconitase.
The control of cellular iron homeostasis involves the coordinate post-transcriptional regulation of ferritin mRNA translation and transferring receptor mRNA stability. These regulatory events are mediated by a soluble cytoplasmic protein, iron regulatory factor (IRF), which binds specifically to mRNA hairpin structures, termed iron-responsive elements (IREs), in the respective mRNAs. IRF is modulated by variations of cellular iron levels and exists as either an apo-protein or a [4Fe-4S]-cluster protein. The two conformations show distinct, mutually exclusive functions. High-affinity IRE binding is observed with the apo-form induced by iron deprivation, but is lost under high iron conditions when IRF is converted to the [4Fe-4S]-cluster form which shows cytoplasmic aconitase activity. Moreover, IRE binding is inactivated by the sulfhydryl-oxidizing agent diamide and fully activated in vitro by 2% 2-mercapto-ethanol, whereas alkylation of IRF inhibits IRE binding. In the present study, we analyzed each of the above features using site-directed mutants of recombinant human IRF. The results support the bifunctional nature of IRF. We conclude that cysteines 437, 503 and 506 anchor the [4Fe-4S]-cluster, and are essential to the aconitase activity. Mutagenesis changing any of the cysteines to serine leads to constitutive RNA binding in 0.02% 2-mercaptoethanol. Cysteine 437 is particularly critical to the RNA-protein interaction. The spontaneous or diamide-induced formation of disulfide bonds between cysteines 437 and 503 or 437 and 506, in apo-IRF, as well as its alkylation by N-ethylmaleimide, inhibit binding to the IRE.(ABSTRACT TRUNCATED AT 250 WORDS)
Hirling
H
H
Swiss Institute for Experimental Cancer Research (ISREC), Genetics Unit, Epalinges.
Henderson
B R
BR
Kühn
L C
LC
D000154:Aconitate Hydratase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000201:enzymology; D003958:Diamide / Q000494:pharmacology; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D007501:Iron / Q000378:metabolism; D035925:Iron-Regulatory Proteins; D007739:L Cells; D008623:Mercaptoethanol / Q000494:pharmacology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D035925:Iron-Regulatory Proteins; D016601:RNA-Binding Proteins; D003958:Diamide; D008623:Mercaptoethanol; D012313:RNA; D007501:Iron; D000154:Aconitate Hydratase; D003545:Cysteine; D005033:Ethylmaleimide