Iron-dependent degradation of IRP2 requires its C-terminal region and IRP structural integrity.
BACKGROUND
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. However, mutants lacking this fragment remain sensitive to degradation in iron-replete cells.


RESULTS
To 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.


CONCLUSIONS
Our 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.
Wang
Jian
J
Lady Davis Institute for Medical Research, Sir Mortimer B, Davis Jewish General Hospital, Montreal, Quebec, Canada. jian.wang@utsouthwestern.edu
Chen
Guohua
G
Lee
Julie
J
Pantopoulos
Kostas
K
D045744:Cell Line, Tumor; D006801:Humans; D007501:Iron / Q000378:metabolism*; D035941:Iron Regulatory Protein 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D035942:Iron Regulatory Protein 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D054875:Ubiquitination
D007501:Iron; D046988:Proteasome Endopeptidase Complex; D035941:Iron Regulatory Protein 1; D035942:Iron Regulatory Protein 2