In earlier years, the laboratory identified and characterized the cis and trans elements mediating the iron-dependent alterations in the abundance of ferritin and the transferrin receptor. Iron-responsive elements (IREs) are RNA stem-loops found in the 5' end of ferritin mRNA and the 3' end of transferrin receptor mRNA. We have cloned, expressed, and characterized two essential iron-sensing proteins, Iron Regulatory Protein 1 (IRP1) and Iron Regulatory Protein 2 (IRP2). IRPs bind to IREs when iron levels are depleted, resulting in the inhibition of translation of ferritin mRNA and other IRE-containing transcripts and prolongation of the half-life of the transferrin receptor mRNA. IRP1 is an iron-sulfur protein related to mitochondrial aconitase, a citric acid cycle enzyme. It functions as a cytosolic aconitase in cells that are iron replete. Regulation of RNA-binding activity of IRP1 involves a transition from a form of IRP1 in which a [4Fe-4S] cluster is bound to a form that loses both iron and aconitase activity. The [4Fe-4S]-containing protein does not bind to IREs. Controlled degradation of the iron-sulfur cluster and mutagenesis reveals that the physiologically relevant form of the RNA binding protein in iron-depleted cells is apoprotein. The status of the cluster appears to determine whether IRP1 will bind RNA.

The Molecular Basis of Intracellular Iron Metabolism
Recently, we have identified mammalian enzymes of iron-sulfur cluster assembly that are homologous to the NifS and Nif U genes implicated in bacterial iron-sulfur cluster assembly, and we have shown that these gene products facilitate assembly of the iron- sulfur cluster of IRP1. IRP2 also binds to IREs in iron-depleted cells, but, unlike IRP1, IRP2 is degraded in cells that are iron replete. Experimental evidence indicates that IRP2 binds to iron and undergoes iron-catalyzed oxidation. The oxidized protein is then selectively ubiquitinated and degraded by the proteasome. Indirect evidence suggests that numerous other proteins are degraded by a pathway in which oxidative modification is followed by ubiquitination and proteasomal degradation of the ubiquitinated substrate. To approach questions about the physiology of iron metabolism, loss of function mutations of IRP1 and IRP2 have been generated in mice through homologous recombination in embryonic cell lines. In the absence of provocative stimuli, there are no abnormalities in iron metabolism associated with loss of IRP1 function. IRP2-/-mice develop a progressive movement disorder characterized by gait abnormalities and tremor. Animals develop axonal degeneration characterized by iron accumulation in axons. Ferritin overexpression occurs in affected neurons, and ferritin accumulation occurs in axons. Affected cells are those that depend mainly on IRP2 for repression of ferritin translation. Animals that lack both IRP1 and IRP2 are embryonic lethal. Blastocyst harvest may yield cells that completely lack IRPs.