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.