Abstract
Iron-sulfur cluster-dependent interconversion of iron regulatory protein 1 (IRP1) between its RNA binding and cytosolic aconitase (c-acon) forms controls vertebrate iron homeostasis. Cluster removal from c-acon is thought to include oxidative demetallation as a required step, but little else is understood about the process of conversion to IRP1. In comparison with c-acon(WT), Ser(138) phosphomimetic mutants of c-acon contain an unstable [4Fe-4S] cluster and were used as tools to further define the pathway(s) of iron-sulfur cluster disassembly. Under anaerobic conditions cluster insertion into purified IRP1(S138E) and cluster loss on treatment with NO regulated aconitase and RNA binding activity over a similar range as observed for IRP1(WT). However, activation of RNA binding of c-acon(S138E) was an order of magnitude more sensitive to NO than for c-acon(WT). Consistent with this, an altered set point between RNA-binding and aconitase forms was observed for IRP1(S138E) when expressed in HEK cells. Active c-acon(S138E) could only accumulate under hypoxic conditions, suggesting enhanced cluster disassembly in normoxia. Cluster disassembly mechanisms were further probed by determining the impact of iron chelation on acon activity. Unexpectedly EDTA rapidly inhibited c-acon(S138E) activity without affecting c-acon(WT). Additional chelator experiments suggested that cluster loss can be initiated in c-acon(S138E) through a spontaneous nonoxidative demetallation process. Taken together, our results support a model wherein Ser(138) phosphorylation sensitizes IRP1/c-acon to decreased iron availability by allowing the [4Fe-4S](2+) cluster to cycle with [3Fe-4S](0) in the absence of cluster perturbants, indicating that regulation can be initiated merely by changes in iron availability.
Highlights
Generation of iron-responsive elements (IREs) RNA binding activity from c-acon requires complete removal of the iron-sulfur cluster along with reduction of critical Cys [8]
In this study we have investigated the impact of cluster perturbants and iron chelators on the capacity for cluster-dependent regulation of Ser138 phosphomimetic mutants of iron regulatory protein 1 (IRP1)
A Ser138 Phosphomimetic Mutant of IRP1 Is Regulated by the Iron-Sulfur Switch in Vitro—Previous studies had suggested that Ser138 phosphorylation might completely uncouple IRP1 from the iron-sulfur switch mechanism, allowing for its regulation only through iron-mediated protein degradation [22]
Summary
Generation of IRE RNA binding activity from c-acon requires complete removal of the iron-sulfur cluster along with reduction of critical Cys [8]. Ser138 phosphomimetic mutants of IRP1 (IRP1S138D and IRP1S138E) can assemble a [4Fe-4S] cluster and exhibit aconitase activity as the purified protein or when supporting aconitase-dependent growth in yeast in vivo. In both cases the iron-sulfur cluster displayed markedly increased sensitivity to oxygen or reactive oxygen species and altered accumulation of a likely intermediate in the conversion of c-acon to IRP1 [10, 21]. The capacity of both IRP1S138D and IRP1S138E to support aconitase-dependent growth in yeast was more sensitive to iron depletion than that of IRP1WT, implying that the phosphomimetic mutants exhibit increased rates of cluster removal in vivo [21]. Our results support a model wherein Ser138 phosphorylation allows for regulation of IRP1 via the iron-sulfur switch mechanism in a manner that enhances responsiveness to changes of intracellular iron status or oxidative/nitrosative stress while preventing potentially lethal overaccumulation of IRP1 through iron-mediated degradation of the protein
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
