In this proposal, the genes and proteins involved in trafficking of iron between the plasma membrane and protein within mitochondria of Saccharomyces cerevisiae will be defined. A novel collection of yeast mutants will be studied that exhibit constitutively high cellular iron uptake, iron sensitivity and accumulation of iron within mitochondria. These mutants will be organized into complementation groups, the genes(s) will be cloned and the gene products(s) characterized. Mutants of this type, such as the ma15 mutant, accumulate iron as an iron-protein complex with mitochondria, and this complex will be purified and characterized in terms of its protein and iron composition. Furthermore, a specific iron protein isolated from mitochondria appeared deficient in the ma15 mutant (33 kDa iron protein). This protein will be purified and identified. The molecular defect in these mutants may be directly or indirectly related to this protein, and it will be important to ascertain if the lack of this iron protein represents a general defect in iron delivery to proteins within mitochondria or a more narrow defect. The second specific aim is to determine the biochemical features of iron import into mitochondria. The rates of import into mitochondria will be determined for living cells and/or isolated mitochondria, looking for abnormalities that might be specific for the mutants. Methods developed for measuring protein import into purified mitochondria will be used to aid in developments of these studies. Finally, specific aim 3 is aimed at determining the nature of iron toxicity in the ma15 mutant. The basis of the small colony phenotype and iron sensitivity will be evaluated and the evidence that mtDNA represents a target for toxic iron damage in these mutants will be evaluated using methods unique to yeast. The yeast mutants that accumulate iron in dense deposits within mitochondria while failing to properly synthesize iron proteins bear a resemblance to the erythroid progenitors of a group of human disorders called sideroblastic anemia. The acquired form of sideroblastic anemia often represents a preleukemic state, and the basis for the disease and for the transition to leukemia is unknown, Thus, clinical relevance of the yeast work will be sought by looking for conserved genes and function.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Badman, David G
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Pennsylvania
Internal Medicine/Medicine
Schools of Medicine
United States
Zip Code
Rocha, Agostinho G; Knight, Simon A B; Pandey, Alok et al. (2018) Cysteine desulfurase is regulated by phosphorylation of Nfs1 in yeast mitochondria. Mitochondrion 40:29-41
Dzul, Stephen P; Rocha, Agostinho G; Rawat, Swati et al. (2017) In vitro characterization of a novel Isu homologue from Drosophila melanogaster for de novo FeS-cluster formation. Metallomics 9:48-60
Rocha, Agostinho G; Knight, Simon A B; Pandey, Alok et al. (2017) Nfs1 cysteine desulfurase protein complexes and phosphorylation sites as assessed by mass spectrometry. Data Brief 15:775-799
Pain, Debkumar; Dancis, Andrew (2016) Roles of Fe-S proteins: from cofactor synthesis to iron homeostasis to protein synthesis. Curr Opin Genet Dev 38:45-51
Rodrigues, Andria V; Kandegedara, Ashoka; Rotondo, John A et al. (2015) Iron loading site on the Fe-S cluster assembly scaffold protein is distinct from the active site. Biometals 28:567-76
Cook, Jeremy D; Kondapalli, Kalyan C; Rawat, Swati et al. (2010) Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly. Biochemistry 49:8756-65
Amutha, Boominathan; Gordon, Donna M; Dancis, Andrew et al. (2009) Chapter 14 Nucleotide-dependent iron-sulfur cluster biogenesis of endogenous and imported apoproteins in isolated intact mitochondria. Methods Enzymol 456:247-66
Kondapalli, Kalyan C; Kok, Nicole M; Dancis, Andrew et al. (2008) Drosophila frataxin: an iron chaperone during cellular Fe-S cluster bioassembly. Biochemistry 47:6917-27
Zhang, Yan; Lyver, Elise R; Nakamaru-Ogiso, Eiko et al. (2008) Dre2, a conserved eukaryotic Fe/S cluster protein, functions in cytosolic Fe/S protein biogenesis. Mol Cell Biol 28:5569-82
Amutha, Boominathan; Gordon, Donna M; Gu, Yajuan et al. (2008) GTP is required for iron-sulfur cluster biogenesis in mitochondria. J Biol Chem 283:1362-71

Showing the most recent 10 out of 29 publications