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Iron-dependent degradation of IRP2 requires its C-terminal region and IRP structural integrity.

Wang J, Chen G, Lee J, Pantopoulos K - BMC Mol. Biol. (2008)

Bottom Line: However, the fusion of C-terminal IRP2 fragments to luciferase failed to sensitize the indicator protein for degradation in iron-loaded cells.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lady Davis Institute for Medical Research, Sir Mortimer B, Davis Jewish General Hospital, Montreal, Quebec, Canada. jian.wang@utsouthwestern.edu

ABSTRACT

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.

Conclusion: 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.

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Truncation of the C-terminal domain 4 stabilizes IRP2 against iron. (A) Schematic representation of the deletion mutants showing domains 1–4, the hinge which links domains 3 and 4, and the C-terminal HA tag. (B-F) H1299 cells engineered to express the above mutants (in two or three independent clones) were treated overnight (14 h) with 30 μg/ml FAC and lysates were subjected to Western blotting with HA (top) and β-actin (bottom) antibodies. The different migration of wild type IRP2 and the ΔC60 and ΔC168 deletion mutants is illustrated in (B).
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Figure 5: Truncation of the C-terminal domain 4 stabilizes IRP2 against iron. (A) Schematic representation of the deletion mutants showing domains 1–4, the hinge which links domains 3 and 4, and the C-terminal HA tag. (B-F) H1299 cells engineered to express the above mutants (in two or three independent clones) were treated overnight (14 h) with 30 μg/ml FAC and lysates were subjected to Western blotting with HA (top) and β-actin (bottom) antibodies. The different migration of wild type IRP2 and the ΔC60 and ΔC168 deletion mutants is illustrated in (B).

Mentions: The above data are consistent with an involvement of IRP2 domain 4 in tagging the protein for proteasomal degradation. To identify minimal requirements for this activity, further deletion mutants within domain 4 were generated, expressed in H1299 cells and analyzed for their response to iron (Fig. 5). We first noticed that the ΔC168, but also the ΔC60 deletion mutants remained stable in iron-loaded cells (Figs. 5B–D), indicating that the sequence encompassing the 60 C-terminal amino acids of IRP2 may possess iron-sensing properties. To further address this, we generated IRP2 variants lacking either the 34 C-terminal (ΔC34) or the adjacent 26 amino acids (ΔC26). Both the ΔC34, as well as the ΔC26 deletion mutants were resistant to iron-mediated degradation (Figs. 5E and 5F).


Iron-dependent degradation of IRP2 requires its C-terminal region and IRP structural integrity.

Wang J, Chen G, Lee J, Pantopoulos K - BMC Mol. Biol. (2008)

Truncation of the C-terminal domain 4 stabilizes IRP2 against iron. (A) Schematic representation of the deletion mutants showing domains 1–4, the hinge which links domains 3 and 4, and the C-terminal HA tag. (B-F) H1299 cells engineered to express the above mutants (in two or three independent clones) were treated overnight (14 h) with 30 μg/ml FAC and lysates were subjected to Western blotting with HA (top) and β-actin (bottom) antibodies. The different migration of wild type IRP2 and the ΔC60 and ΔC168 deletion mutants is illustrated in (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2267205&req=5

Figure 5: Truncation of the C-terminal domain 4 stabilizes IRP2 against iron. (A) Schematic representation of the deletion mutants showing domains 1–4, the hinge which links domains 3 and 4, and the C-terminal HA tag. (B-F) H1299 cells engineered to express the above mutants (in two or three independent clones) were treated overnight (14 h) with 30 μg/ml FAC and lysates were subjected to Western blotting with HA (top) and β-actin (bottom) antibodies. The different migration of wild type IRP2 and the ΔC60 and ΔC168 deletion mutants is illustrated in (B).
Mentions: The above data are consistent with an involvement of IRP2 domain 4 in tagging the protein for proteasomal degradation. To identify minimal requirements for this activity, further deletion mutants within domain 4 were generated, expressed in H1299 cells and analyzed for their response to iron (Fig. 5). We first noticed that the ΔC168, but also the ΔC60 deletion mutants remained stable in iron-loaded cells (Figs. 5B–D), indicating that the sequence encompassing the 60 C-terminal amino acids of IRP2 may possess iron-sensing properties. To further address this, we generated IRP2 variants lacking either the 34 C-terminal (ΔC34) or the adjacent 26 amino acids (ΔC26). Both the ΔC34, as well as the ΔC26 deletion mutants were resistant to iron-mediated degradation (Figs. 5E and 5F).

Bottom Line: However, the fusion of C-terminal IRP2 fragments to luciferase failed to sensitize the indicator protein for degradation in iron-loaded cells.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lady Davis Institute for Medical Research, Sir Mortimer B, Davis Jewish General Hospital, Montreal, Quebec, Canada. jian.wang@utsouthwestern.edu

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus