Abstract
BackgroundIron regulatory protein 2 (IRP2), a post-transcriptional regulator of cellular iron metabolism, undergoes iron-dependent degradation via the ubiquitin-proteasome pathway. A stretch of 73 amino acids within the N-terminal domain 1 of the protein was reported to function as an iron sensor. However, mutants lacking this fragment remain sensitive to degradation in iron-replete cells.ResultsTo identify elements within IRP2 involved in the control of its stability, we undertook a systematic mutagenesis approach. Truncated versions of IRP2 were expressed in H1299 cells and analyzed for their response to iron. Deletion mutants lacking the entire C-terminal domain 4 (amino acids 719–963) of IRP2 remained stable following iron treatments. Moreover, the replacement of domain 4 of IRP1 with the corresponding region of IRP2 sensitized the chimerical IRP11–3/IRP24 protein to iron-dependent degradation, while the reverse manipulation gave rise to a stable chimerical IRP21–3/IRP14 protein. The deletion of just 26 or 34 C-terminal amino acids stabilized IRP2 against iron. However, the fusion of C-terminal IRP2 fragments to luciferase failed to sensitize the indicator protein for degradation in iron-loaded cells.ConclusionOur data suggest that the C-terminus of IRP2 contains elements that are necessary but not sufficient for iron-dependent degradation. The functionality of these elements depends upon the overall IRP structure.
Highlights
Iron regulatory protein 2 (IRP2), a post-transcriptional regulator of cellular iron metabolism, undergoes iron-dependent degradation via the ubiquitin-proteasome pathway
IRPs regulate the expression of additional iron responsive elements (IREs)-containing transcripts, such as those encoding erythroid aminolevulinate synthase (ALAS2), mitochondrial aconitase, the iron transporter ferroportin 1, myotonic dystrophy kinaserelated Cdc42-binding kinase α (MRCK α), hypoxia inducible factor 2 α (HIF2α), and splice variants of the divalent metal transporter DMT1 and the kinase Cdc14A [2,3,4]
The C-terminal domain of IRP2 is necessary for its irondependent degradation Even though the crystal structure of IRP2 has not yet been solved, on the basis of IRP1 structural data [6,7] and sequence homology between IRP1 and IRP2, it can be predicted that the IRP2 molecule is composed of three compact domains (1–3) linked to a fourth domain via a hinge region (Fig. 1A)
Summary
Iron regulatory protein 2 (IRP2), a post-transcriptional regulator of cellular iron metabolism, undergoes iron-dependent degradation via the ubiquitin-proteasome pathway. A stretch of 73 amino acids within the N-terminal domain 1 of the protein was reported to function as an iron sensor Mutants lacking this fragment remain sensitive to degradation in ironreplete cells. In iron-replete cells, IRP1 binds a cubane 4Fe-4S cluster, which precludes IRE-binding, renders the protein to a cytosolic aconitase and maintains it in a closed conformation [6,7]. Under these conditions, IRP2 undergoes rapid ubiquitination and degradation by the proteasome [1,2,5]. Phosphorylation or defects in Fe-S cluster assembly may sensitize IRP1 to iron-dependent proteasomal degradation, albeit with slower kinetics compared to IRP2 [8,9,10]
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