Disruption of the iron homeostasis dynamic process originates significant damage in cells. Therefore, organisms and yeast cells in particular have evolved sophisticated mechanisms that, on one hand avoid the drastic consequences of iron scarcity and, on the other hand circumvent the toxic effects of iron overload. It is relevant to understand how cells cope with high Fe levels, since hemachromatosis caused by Fe overload is one of the most common human genetic diseases.
The work here reported shows that, as transcriptional primary response to iron excess, S. cerevisiae
represses genes involved in iron uptake, FET3
, as well as in iron mobilization from the vacuole, SMF3
; it also induces genes implicated in Fe storage, CCC1
, and in Aft1 export from the nucleus, GRX4
. Apparently, the alteration in the expression pattern of these genes is enough to control the intracellular iron concentration that would lead to Fe-induced toxicity. CCC1
induction as well as FET3
repression, when in the presence of high-Fe, have been also reported by other authors 
. Surprisingly, only three genes of the Fe-regulon were found to be downregulated (see , FET3
). Because under iron overload conditions Aft1 is exported from the nucleus, one would expect to see a greater number of Aft1-dependent genes being downregulated. Yun et al.
noticed that Aft1-dependent transcription is repressed in Fe-sufficient medium 
. Furthermore, under these conditions, RNAse III Rnt1-mediated RNA surveillance is required to prevent iron toxicity 
. Rnt1 degrades several Aft1-dependent targets, but not FET3
mRNAs; this may explain why we did not detect other Aft1 targets.
The novelty of our analysis is the induction of GRX4
in a Fe-dependent manner. The physiological function of Grx4/3 has been extensively studied and its role in Aft1 activity inhibition was proposed to be due to the sequester of this transcription factor in the cytosol under iron overload conditions 
. Hence, it is likely that cells increase Grx4 levels when facing high-Fe concentrations in order to guarantee that Aft1 is efficiently removed from the nucleus. Grx4/3 may also participate in intracellular iron trafficking, as the double mutant grx4grx3
elicits severe defects in the maturation of cellular Fe-S proteins, heme-containing and di-iron enzymes, despite the constitutive Aft1 activation and the consequent cytosolic iron accumulation 
. Shakoury-Elizeh et al.
observed that under high-Fe pathways involving biotin biosynthesis and nitrogen assimilation via glutamate synthase (Glt1) were activated 
. These pathways requiring Fe-dependent enzymes may possibly lead to the increase of Grx4 levels that correlates with the shift to a most Fe-consuming metabolism. Grx3 and Grx4 bind a Fe-S center together with gluthatione which is crucial for their function 
. Therefore another possibility is that Grx4, itself, may buffer the increased cytosolic iron concentrations under such conditions.
Under iron overload conditions, the transcription factor Yap5 is activated and increases the expression of CCC1
that transports iron into the vacuole, leading to the consequent decrease of the cytosolic iron pool 
. We have now shown that although Yap5 does indeed regulate CCC1
transcription, another yet unidentified Fe-responsive factor drives the expression of CCC1
up to sufficient levels to overcome Fe toxicity. The putative existence of another Fe-responsive factor could justify the almost-normal growth displayed by the yap5
strain in high-Fe medium, as well as the rescue of the poor-growth phenotype of the ccc1
strain by a plasmid harboring the CCC1
gene without YREs (). Furthermore, we showed that, under these conditions, in addition to CCC1
, Yap5 is also regulating the expression of GRX4
(). Consistently with Grx4 dependence on Yap5, we demonstrated, using different approaches, that Yap5 affects Aft1 localization under Fe overload: yap5
cells have more Aft1 in the nucleus and consequently exhibit an upregulation of FET3
(). We cannot however exclude the possibility that Yap5 might as well play a role in iron trafficking, or in buffering iron excess, due to the recent findings on the role of glutaredoxins Grx3/Grx4 in iron metabolism 
Moreover, we demonstrated that Yap5 also plays a role under Fe-adequate growth environments, as in such condition, Yap5 is the major regulator of CCC1 (). As such, in the yap5 mutant strain, CCC1 levels are severely compromised, leading to an increase of cytosolic iron with the consequent inhibition of Aft1 (that localizes mainly in the cytosol) and the concomitant downregulation of its target FET3 ().
In light of the data described herein, we propose that under Fe-adequacy Yap5 is activated leading to the upregulation of CCC1 with the consequent accumulation of Fe in the vacuole and deprivation of the cytosolic iron pool. As a result, Aft1 translocates to the nucleus and upregulates iron uptake genes. Under Fe overload conditions, Yap5 transactivation potential increases leading to the upregulation of GRX4 gene and thus inhibiting Aft1 nuclear localization. Simultaneously, it seems that another iron responsive factor is activated, contributing to the CCC1 induction and most probably bypassing Yap5 regulation (). Further work is in progress in order to clarify the mechanism that controls the Yap5-independent regulation of CCC1.
Model for the hypothetical role of Yap5 in iron metabolism.