Overexpression of iron-responsive element-binding protein and its analytical characterization as the RNA-binding form, devoid of an iron-sulfur cluster.
The iron-responsive element-binding protein (IRE-BP) has been defined and identified as an RNA-binding protein found in iron-deprived eukaryotic cells. IRE-BP binds to stem-loop structures, iron-responsive elements (IREs), which are located in the untranslated regions of the mRNAs for several genes including ferritin, and the transferrin receptor. When bound, IRE-BP prevents ferritin translation and stabilizes the transferrin receptor transcript. When cells are iron replete, an iron-sulfur cluster is ligated to the IRE-BP, the protein loses RNA binding properties, and it acquires aconitase activity. Cytosolic aconitase from liver can be converted into the IRE-BP by oxidative removal of its Fe-S cluster. We describe here overexpression of IRE-BP in baculovirus-infected insect cells which yields IRE-BP devoid of an iron-sulfur cluster. We describe a one-step purification of the IRE-BP and a quantitative analysis of Fe, S2-, S0, protein, and enzyme activity on IRE-BP, as obtained in cell lysates, after purification, and after reconstitution to active aconitase. On the average not more than 3% of the over-expressed purified protein contained an intact Fe-S cluster, and it was demonstrated that that cluster was not lost during purification. Scatchard analysis of RNA-binding data was compatible with a single high-affinity RNA-binding form of the IRE-BP. Active aconitase could be reconstituted from the purified IRE-BP obtained from the expression system by addition of iron, thiol, and sulfide, and the characteristic epr spectrum of the 3Fe form of cytosolic aconitase was obtained after ferricyanide oxidation of the reconstituted material.
Basilion
J P
JP
National Institutes of Health, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Kennedy
M C
MC
Beinert
H
H
Massinople
C M
CM
Klausner
R D
RD
Rouault
T A
TA
D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005293:Ferritins / Q000096:biosynthesis / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007313:Insecta; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D005293:Ferritins