Isolation, characterization, and functional studies of rat liver iron regulatory protein 1.
Ferritin mRNAs are translationally regulated by the binding of either of two cytosolic proteins, iron regulatory protein 1 (IRP1) or IRP2, to the iron responsive element (IRE) located in their 5' untranslated region (UTR). Rat liver IRP1 was purified by anion exchange, gel filtration, and affinity chromatography using a concatemerized version of the IRE. Two bands with M(r) of 95,000 and 100,000 were observed by reducing SDS-PAGE. A single protein was responsible for both bands since: (1) [32P]IRE RNA specifically cross-linked to both components; (2) alkylation with iodoacetamide resulted in formation of a single species with M(r) of 95,000; and (3) they possessed identical peptide patterns after digestion with cyanogen bromide. The N-terminal sequence of rat liver IRP1 was MKNPFAHLAEPLDPAQPGKKFNLNKLEDSRYGRLPFXIRVLLEAAV which is identical to the sequence deduced from the cDNA. Rat liver IRP1 has an amino acid composition similar to that of bovine liver caconitase. Several species of IRP1 were observed by two-dimensional gel electrophoresis with pIs ranging from 7.5 to 8.0. Rat liver IRP1 bound the IRE with high affinity (K(D) = 0.04 nM) and repressed translation of ferritin mRNA in vitro. IRP1 bound 100-fold less well to an IRE variant and failed to significantly repress translation of a ferritin mRNA containing the mutated IRE. We conclude that decreases in the affinity of interaction between IRP1 and the IRE, of a magnitude similar to that observed when the binding protein in converted to c-aconitase, are sufficient to significantly enhance translation of ferritin mRNA in vitro.
Eisenstein
R S
RS
Department of Nutritional Sciences, University of Wisconsin, Madison 53706, USA. eisenste@nutrisci.wisc.edu
Barton
H A
HA
Pettingell
W H
WH
Bomford
A B
AB
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005293:Ferritins / Q000235:genetics / Q000378:metabolism; D035941:Iron Regulatory Protein 1; D035942:Iron Regulatory Protein 2; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007526:Isoelectric Point; D008099:Liver / Q000737:chemistry*; D008969:Molecular Sequence Data; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats
D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D005293:Ferritins; D035941:Iron Regulatory Protein 1; D035942:Iron Regulatory Protein 2