Iron-dependent degradation of apo-IRP1 by the ubiquitin-proteasome pathway.
Iron regulatory protein 1 (IRP1) controls the translation or stability of several mRNAs by binding to "iron-responsive elements" within their untranslated regions. In iron-replete cells, IRP1 assembles a cubane iron-sulfur cluster (ISC) that inhibits RNA-binding activity and converts the protein to cytosolic aconitase. We show that the constitutive IRP1(C437S) mutant, which fails to form an ISC, is destabilized by iron. Thus, exposure of H1299 cells to ferric ammonium citrate reduced the half-life of transfected IRP1(C437S) from approximately 24 h to approximately 10 h. The iron-dependent degradation of IRP1(C437S) involved ubiquitination, required ongoing transcription and translation, and could be efficiently blocked by the proteasomal inhibitors MG132 and lactacystin. Similar results were obtained with overexpressed wild-type IRP1, which predominated in the apo-form even in iron-loaded H1299 cells, possibly due to saturation of the ISC assembly machinery. Importantly, inhibition of ISC biogenesis in HeLa cells by small interfering RNA knockdown of the cysteine desulfurase Nfs1 sensitized endogenous IRP1 for iron-dependent degradation. Collectively, these data uncover a mechanism for the regulation of IRP1 abundance as a means to control its RNA-binding activity, when the ISC assembly pathway is impaired.
Wang
Jian
J
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, 3755 Cote-Ste-Catherine Road, Montreal, Quebec H3T 1E2, Canada.
Fillebeen
Carine
C
Chen
Guohua
G
Biederbick
Annette
A
Lill
Roland
R
Pantopoulos
Kostas
K
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D013437:Carbon-Sulfur Lyases / Q000378:metabolism; D045744:Cell Line, Tumor; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006801:Humans; D007501:Iron / Q000378:metabolism*; D035941:Iron Regulatory Protein 1 / Q000235:genetics / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D053858:Metabolic Networks and Pathways; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D014176:Protein Biosynthesis / Q000502:physiology; D015533:Transcriptional Activation / Q000502:physiology; D025801:Ubiquitin / Q000378:metabolism*
D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D061988:Proteasome Inhibitors; D025801:Ubiquitin; C067713:lactacystin; D007501:Iron; D046988:Proteasome Endopeptidase Complex; D035941:Iron Regulatory Protein 1; D013437:Carbon-Sulfur Lyases; C513492:NFS1 protein, human; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine