Novel role of phosphorylation in Fe-S cluster stability revealed by phosphomimetic mutations at Ser-138 of iron regulatory protein 1

Abstract

Animals regulate iron metabolism largely through the action of the iron regulatory proteins (IRPs). IRPs modulate mRNA utilization by binding to iron-responsive elements (IRE) in the 5' or 3' untranslated region of mRNAs encoding proteins involved in iron homeostasis or energy production. IRP1 is also the cytosolic isoform of aconitase. The activities of IRP1 are mutually exclusive and are modulated through the assembly/disassembly of its [4Fe-4S] cluster, reversibly converting it between an IRE-binding protein and cytosolic aconitase. IRP1 is also phosphoregulated by protein kinase C, but the mechanism by which phosphorylation posttranslationally increases IRE binding activity has not been fully defined. To investigate this, Ser-138 (S138), a PKC phosphorylation site, was mutated to phosphomimetic glutamate (S138E), aspartate (S138D), or nonphosphorylatable alanine (S138A). The S138E IRP1 mutant and, to a lesser extent, the S138D IRP1 mutant were impaired in aconitase function in yeast when grown aerobically but not when grown anaerobically. Purified wild-type and mutant IRP1s could be reconstituted to active aconitases anaerobically. However, when exposed to oxygen, the [4Fe-4S] cluster of the S138D and S138E mutants decayed 5-fold and 20-fold faster, respectively, than was observed for wild-type IRP1. Our findings suggest that stability of the Fe-S cluster of IRP1 can be regulated by phosphorylation and reveal a mechanism whereby the balance between the IRE binding and [4Fe-4S] forms of IRP1 can be modulated independently of cellular iron status. Furthermore, our results show that IRP1 can function as an oxygen-modulated posttranscriptional regulator of gene expression.

Figure 1

Rescue of aco1 yeast from glutamate auxotrophy by expression of S138 IRP1 mutants. Yeast expressing either wild type (WT) or S138 (S138A, S138D, or S138E) IRP1 mutants were grown overnight in SD-Ura, washed twice with sterile H₂O, and suspended into SD-Ura lacking glutamate. Nontransformed yeast (0615d) were grown in SD supplemented with uracil. Ten microliters containing between 2 × 10⁵ and 2 × 10² yeast cells were spotted onto agar plates containing SD supplemented with uracil with or without glutamate as indicated. Growth after 4 days is shown. Columns 1 and 5, 2 × 10⁵ cells; columns 2 and 6, 2 × 10⁴ cells; columns 3 and 7, 2 × 10³ cells; columns 4 and 8, 2 × 10² cells. (a) Aerobic growth. (b) Effect of anaerobiosis on growth of aco1 yeast expressing S138 IRP1 mutants on glutamate-free media. Growth analysis was performed as described above except that plates were incubated in an anaerobic chamber.

Figure 2

Aconitase activity of S138 IRP1 mutants in aerobically prepared cytoplasmic extracts of aco1 yeast. Yeast expressing the indicated IRP1 mutants were grown to mid-logarithmic phase, harvested, and cytoplasmic extracts were prepared and analyzed for aconitase activity. Activities shown are per mg of total extracted protein and are an average of three independent experiments. Error bars indicate SD.

Figure 3

Half-life of aconitase activity of S138 IRP1 mutants upon exposure to oxygen. IRP1 mutants were overexpressed and purified from yeast and subjected to in vitro cluster reconstitution anaerobically. Aconitase activity was measured at various times after diluting the reconstituted protein into air-saturated buffer. The half-life (t_1/2) of aconitase activity for each IRP1 mutant is indicated in the inset.