Iron Trafficking and Regulation in Saccaromyces cerevisiae: The Role of Frataxin and the Involvement of Mitochondria in Cytosolic Iron Sulfur Cluster Biosynthesis

Abstract

Iron-sulfur-cluster (ISC) assembly occurs in both mitochondria and cytosol. Defects in mitochondrial ISC assembly can result in diseases such as Friedrich’s Ataxia which results from a deficiency in frataxin. The mechanism of disease development and the biochemical role of frataxin in that process are uncertain. Mitochondrial ISC assembly occurs independently of the cytosolic process, while cytosolic assembly depends on mitochondria. This asymmetry likely arises from the use of an exported low-molecular-mass (LMM) species from mitochondria in cytosolic ISC assembly; however, no such species has been directly detected.

The research described in this dissertation addresses both problems. To address the role of frataxin in the development of Friedreich’s ataxia, a strain of S. cerevisiae in which expression of the yeast frataxin homologue (Yfh1) could be adjusted was characterized using biochemical, biophysical and computational approaches. Mössbauer spectroscopy was used to investigate the iron content of mutant cells under a variety of Yfh1 expression levels and growth conditions. In normoxia, mutant cells exhibited slowed growth and iron nanoparticles. However, in hypoxia, the mutant cells grew at rates similar to WT cells, had similar iron content, and were dominated by high-spin FeII rather than FeIII nanoparticles. Mitochondria from mutant hypoxic cells contained similar levels of ISCs to WT cells, confirming that Yfh1 was unnecessary for ISC assembly. These results informed a mathematical model of iron trafficking and regulation in cells that could simulate disease progression.

To address the relationship between mitochondrial and cytosolic ISC assembly processes, an assay was developed to investigate iron export from mitochondria for use in the cytosolic iron sulfur cluster assembly. Mitochondria were isolated from 57Fe-enriched cells and incubated in buffers that activated ISC assembly. After incubation, mitochondria were separated from supernatant, and samples were run on a novel liquid chromatography system coupled to an ICP-MS. When activated, some mitochondrial iron was imported into mitochondrial proteins. Additionally, activated mitochondria exported two iron complexes. When mitochondria were mixed with isolated cytosol and activated, multiple cytosolic iron-containing proteins became enriched with mitochondrial iron. The results from both studies contribute to the understanding of iron trafficking and regulation in eukaryotic cells and may eventually contribute to a better understanding and treatments for iron-related diseases.