Rolle der cytosolischen Glutaredoxine im zellulären Eisenmetabolismus in Eukaryoten

Glutaredoxins are found in all phylogenetic kingdoms of life. This protein family includes dithiol-glutaredoxins that contain a CPYC active site and are involved in oxidative stress protection. Single domain glutaredoxins with a CGFS active site play crucial roles in the maturation of cellular iron-sulfur (Fe/S) proteins and are usually localized in mitochondria or chloroplasts. Multidomain monothiol glutaredoxins are fusion proteins that consist of an N-terminal thioredoxin domain and one or more C-terminal glutaredoxin domain(s). This subgroup of glutaredoxins is found in the cytosol and nucleus of nearly all higher eukaryotes. In baker’s yeast S. cerevisiae, the multidomain glutaredoxins Grx3 and Grx4 bind Fe/S cofactors and play a crucial role in the maturation of all classes of iron-containing proteins and the regulation of cellular iron homeostasis. The aim of this work was a refined functional characterization of the multidomain monothiol glutaredoxins and was centered on two major questions. (1) Which structural differences enable the otherwise similar glutaredoxins to each perform a specific function? (2) Is the described function of the S. cerevisiae multidomain glutaredoxins in the iron metabolism conserved in higher eukaryotes? The first question was followed by the analysis of the effects of site-directed mutations on the functionality of Grx4 in S. cerevisiae. The analysis showed that a dithiol, instead of the monthiol active site in Grx4 causes a strong decrease in iron binding and a moderate loss of function in vivo. In contrast to the single domain glutaredoxins, a prolin in the active site of Grx4 is fully compatible with the binding of a Fe/S cluster. Furthermore, the thioredoxin domain is essential for function. Most likely, this domain serves as a binding platform for other proteins. Yeast Grx3 and Grx4 interact with the iron-responsive transcription factor Aft1 and inactivate Aft1 under iron sufficiency. Here it could be shown that the regulation of Aft1 is incompatible with a dithiol active site of Grx4 and that the C terminus of Grx4 serves as the binding site for Aft1. Complex formation between Grx4 and Aft1 is not iron-dependent and increased when no Fe/S cluster is bound to Grx4. Hence, only the inactivation of Aft1, not the binding to Grx4 depends on the Fe/S cluster on Grx4.Collectively, this work identified structural requirements for the function of the multidomain monthiol glutaredoxins in the cellular iron metabolism of S. cerevisiae. To answer the second question, the effects of siRNA-mediated depletion of Grx3 in HeLa cells were analysed. Similar to yeast cells, Grx3-depleted HeLa cells showed defects in the assembly of cytosolic and nuclear Fe/S proteins. A defect in the maturation of the “Iron Regulatory Protein 1” (IRP1) caused the degradation of the apo-form of this protein and the subsequent effects on the cellular iron metabolism in Grx3-depleted HeLa cells. In contrast to yeast cells, Grx3 depletion in HeLa cells showed no effects on the assembly of mitochondrial Fe/S Cluster- or heme-depending enzymes. Furthermore, despite an altered iron metabolism, the transcriptome of Grx3-depleted HeLa cells showed no alterations in the regulation of typical iron-responsive genes that have a function in mitochondria or under hypoxic conditions. This observation and the missing effects on the mitochondrial iron-depending enzymes are conspicuous differences to yeast. Collectively, this work confirms a conserved function of Grx3 regarding the biogenesis of cytosolic Fe/S proteins, but not for mitochondrial iron-depending proteins in higher eukaryotes.