Abstract
The transcription factors Aft1 and Aft2 from Saccharomyces cerevisiae regulate the expression of genes involved in iron homeostasis. These factors induce the expression of iron regulon genes in iron-deficient yeast but are inactivated in iron-replete cells. Iron inhibition of Aft1/Aft2 was previously shown to be dependent on mitochondrial components required for cytosolic iron sulfur protein biogenesis. We presently show that the nuclear monothiol glutaredoxins Grx3 and Grx4 are critical for iron inhibition of Aft1 in yeast cells. Cells lacking both glutaredoxins show constitutive expression of iron regulon genes. Overexpression of Grx4 attenuates wild type Aft1 activity. The thioredoxin-like domain in Grx3 and Grx4 is dispensable in mediating iron inhibition of Aft1 activity, whereas the conserved cysteine that is part of the conserved CGFS motif in monothiol glutaredoxins is essential for this function. Grx3 and Grx4 interact with Aft1 as shown by two-hybrid interactions and co-immunoprecipitation assays. The interaction between glutaredoxins and Aft1 is not modulated by the iron status of cells but is dependent on the conserved glutaredoxin domain Cys residue. Thus, Grx3 and Grx4 are novel components required for Aft1 iron regulation that most likely occurs in the nucleus.
Highlights
Aft1 is localized to the nucleus under low iron conditions and to cytoplasm under iron-sufficient conditions [9]
The growth medium was supplemented with 0.1 mM bathophenanthroline sulfonate (BPS) as a ferrous iron chelator to lower the availability of iron or supplemented with 0.1 mM FeCl2
The absence of Grx3 or Grx4 did not affect the full induction of FET3 observed when the iron bioavailability is limited in cells treated with the iron chelator bathophenanthroline sulfonate (BPS)
Summary
Aft is localized to the nucleus under low iron conditions and to cytoplasm under iron-sufficient conditions [9]. It was later shown that disruption of Fe-S cluster biogenesis by diminution in the levels of the cysteine desulfurase (Nfs1) or the frataxin homologue (Yfh1) did not decrease cytosolic iron [13] This is an indication that Aft becomes constitutive due to impairment of a signal created by the mitochondrial Fe-S biosynthetic machinery and not to an indirect effect of alteration in iron compartmentalization. For synthesis of cytosolic and nuclear Fe-S proteins, mitochondria export a still unknown compound via the mitochondrial inner membrane transporter Atm1 [15]. Iron sensing by Aft and Aft requires proper mitochondrial Fe-S cluster biosynthesis as well as a functional export to the cytoplasm. It does not require the CIA machinery [11], demonstrating that iron sensing by Aft1/Aft is not linked to the maturation of cytosolic 4Fe-4S clusters
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