Malini Gupta

Date of Award

Spring 2020

Document Type

Open Access Dissertation


Chemistry and Biochemistry

First Advisor

Caryn E. Outten


As an essential cofactor in a myriad of cellular processes, uptake and mobilization of iron must be tightly controlled. Iron homeostasis in fungi involves balancing iron uptake and storage with iron utilization to achieve adequate, non-toxic levels of this essential nutrient. Extensive work in the non-pathogenic yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have uncovered unique iron regulation networks for each organism that control iron metabolism via distinct molecular mechanisms. The activities of all fungal iron-sensing transcription factors characterized to date are regulated via iron-sulfur cluster signaling.

In Saccharomyces cerevisiae, expression of iron uptake, utilization and storage genes is primarily regulated by transcriptional activators Aft1 (and its paralogue Aft2) and Yap5. Upon iron repletion, a mitochondrially generated Fe-S cluster is delivered to Aft1/Aft2 by a cytosolic complex comprising Grx3/4 and Fra2, leading to the dimerization and eventual export of Aft1/2 to the cytosol. Genetic studies suggest that the protein Fra1 may also have a role in regulating Aft1/2 activity; however, its specific role is unknown. Deletion of either Fra1 or Fra2 in addition to deletion of the vacuolar iron importer Ccc1 restores iron-responsive control of the iron regulon despite the absence of Fra2 or Fra1. Before testing for possible alternate mechanisms for this restoration, we wanted to understand if the mitochondrial iron-sulfur (Fe-S) biogenesis machinery was contributing to inhibition of Aft1/2 in the strains in question. β-galactosidase reporter assay results show that in the absence of a functional mitochondrial iron-sulfur cluster biogenesis machinery (isu1Δ mutants), the iron regulon is constitutively active in all strains except for ccc1Δfra1Δisu1Δ, suggesting that the mitochondria generated inhibitory cluster is not required for inhibition of Aft1 and Aft2 in this mutant strain.

We employed a genetic screen approach to corroborate the in vitro evidence of inhibitory cluster transfer from the cytosolic complex comprising Grx3-Fra2 to Aft1 and Aft2. With the help of error-prone PCR, we successfully created mutated pools of FRA1 and FRA2. Our goal was to isolate point mutations in each of these genes which would result in constitutive inhibition of the iron regulon via Aft1 and Aft2, and once identified, to test the effect of those mutations on inhibitory cluster transfer to Aft1 and Aft2 in vitro. The isolated FRA1 and FRA2 mutants were non-informative non-sense or missense mutations that led to probable defective protein yielding false positive phenotypes in the genetic screen.

Iron homeostasis in Schizosaccharomyces pombe is maintained via transcriptional repression of iron uptake and iron utilization genes. The GATA-type transcriptional repressor Fep1 binds to the promoters of iron uptake and transport genes under iron replete conditions, turning off their expression to avoid iron overload. Fep1 function is controlled at the post-translational level by the cytosolic CGFS glutaredoxin Grx4 and its binding partner Fra2. Biochemical and spectroscopic characterization of Fep1 suggests that it bears a [Fe-S] cofactor and forms a [2Fe-2S]-bound heterocomplex with Grx4 as well as Grx4-Fra2. Monothiol glutaredoxins along with BolA like proteins (Fra2) have been repeatedly shown to be associated with the delivery and transfer of Fe-S clusters to proteins and thus effective communicators of the cellular iron status to iron-responsive transcription factors.

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