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Biophysical Investigation of the Iron in Aft1-1up and Gal-YAH1 Saccharomyces cerevisiae

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Department of Chemistry, Texas A&M University, College Station, Texas 77843-3255, United States
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, United States
*Phone: 979-845-0956. Fax: 979-845-4719. E-mail: [email protected]
Cite this: Biochemistry 2011, 50, 13, 2660–2671
Publication Date (Web):February 28, 2011
Copyright © 2011 American Chemical Society

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    Abstract Image

    Aft1p is a major iron regulator in budding yeast Saccharomyces cerevisiae. It indirectly senses cytosolic Fe status and responds by activating or repressing iron regulon genes. Aft1p within the Aft1-1up strain has a single amino acid mutation which causes it to constitutively activate iron regulon genes regardless of cellular Fe status. This leads to elevated Fe uptake under both low and high Fe growth conditions. Ferredoxin Yah1p is involved in Fe/S cluster assembly, and Aft1p-targeted iron regulon genes are also upregulated in Yah1p-depleted cells. In this study Mössbauer, EPR, and UV−vis spectroscopies were used to characterize the Fe distribution in Aft1-1up and Yah1p-depleted cells. Aft1-1up cells grown in low Fe medium contained more Fe than did WT cells. A basal level of Fe in both WT and Aft1-1up cells was located in mitochondria, primarily in the form of Fe/S clusters and heme centers. The additional Fe in Aft1-1up cells was present as mononuclear HS Fe(III) species. These species are in a nonmitochondrial location, assumed here to be vacuolar. Aft1-1up cells grown in high Fe medium contained far more Fe than found in WT cells. The extra Fe was present as HS Fe(III) ions, probably stored in vacuoles, and as Fe(III) phosphate nanoparticles, located in mitochondria. Yah1p-deficent cells also accumulated nanoparticles in their mitochondria, but they did not contain HS Fe(III) species. Results are interpreted by a proposed model involving three homeostatic regulatory systems, including the Aft1 system, a vacuolar iron regulatory system, and a mitochondrial Fe regulatory system.

    Throughout this paper, we place “phosphate” in parentheses when referring to Fe(III) (phosphate) nanoparticles in Aft1-1up cells because we presume, but have not demonstrated, that they contain phosphate groups. Phosphate is associated with nanoparticles in Atm1p-deficient and Yfh1-deficient mitochondria. (10, 11, 13) Mössbauer spectra of Aft1-1up nanoparticles are nearly indistinguishable from those of Atm1p-deficient cells.

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    The primer pairs used for the RT-PCR experiment (Table S1) and fluorescence data used to determine void volumes in cell pellets (Table S2). This material is available free of charge via the Internet at

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    24. Roland Lill, Bastian Hoffmann, Sabine Molik, Antonio J. Pierik, Nicole Rietzschel, Oliver Stehling, Marta A. Uzarska, Holger Webert, Claudia Wilbrecht, Ulrich Mühlenhoff. The role of mitochondria in cellular iron–sulfur protein biogenesis and iron metabolism. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2012, 1823 (9) , 1491-1508.
    25. Robert Sutak, Alexandra Seguin, Ricardo Garcia‐Serres, Jean‐Louis Oddou, Andrew Dancis, Jan Tachezy, Jean‐Marc Latour, Jean‐Michel Camadro, Emmanuel Lesuisse. Human mitochondrial ferritin improves respiratory function in yeast mutants deficient in iron–sulfur cluster biogenesis, but is not a functional homologue of yeast frataxin. MicrobiologyOpen 2012, 1 (2) , 95-104.
    26. W. Y. Tu, S. Pohl, J. Gray, N. J. Robinson, C. R. Harwood, K. J. Waldron. Cellular Iron Distribution in Bacillus anthracis. Journal of Bacteriology 2012, 194 (5) , 932-940.
    27. Andrew Dancis, Paul A. Lindahl. Iron: Mitochondrial Iron Metabolism and the Synthesis of Iron–Sulfur Clusters. 2004, 1-17.
    28. Caroline C. Philpott, Pamela M. Smith. Iron Starvation Response in Saccharomyces cerevisiae. 2004, 1-16.

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