In most eukaryotic cells (including yeast and humans), the iron insertion step for synthesis of heme occurs exclusively within mitochondria. Likewise, iron insertion for formation of new FeS clusters can occur within mitochondria. Since the mitochondrial inner membrane is impermeable to ions (a requirement for maintaining the electrogenic membrane potential), the question arises of how iron for heme and FeS clusters gets into mitochondria. Recent data from several labs have demonstrated involvement of mitochondrial carrier proteins Mrs3 and Mrs4 in yeast, and orthologs mitoferrin 1 and 2 in vertebrates. However, many aspects of the iron transport process remain unknown. Frataxin a small conserved mitochondrial protein is involved downstream in iron use for heme and FeS clusters within mitochondria, but its precise function is also unclear. Here we propose three genetic and biochemical aims to delve further into this process.
Aim 1) will seek to characterize the iron transport process in isolated yeast mitochondria in short time frames. The role of Mrs3 and Mrs4, and the role of the electrogenic membrane potential in mediating iron transport will be studied. Targeted mutations in the transporters, specifically in the hypothetical substrate binding site, will be evaluated, and the role of a giant protein complex of roughly 660 kDa containing the transporter(s) will be investigated.
Aim 2) will ascertain the role of frataxin in FeS and heme synthesis within mitochondria. The amount of frataxin in mitochondria will be varied over a large range and targeted mutations will be analyzed specifically mutations in interaction sites with iron, with Isu1 (scaffold for FeS cluster assembly) and with ferrochelatase (enzyme for heme synthesis). Assays in isolated mitochondria in short time frames will allow direct effects to be distinguished from secondary effects.
In aim 3), novel genes and proteins connected to iron transport to mitochondria will be sought by means of an ongoing screen to identify mutations that are synthetically lethal with mrs3 and mrs4. Early results show that single mutants (e.g. dre2, tsa1) identified in this screen are involved in iron metabolism. Iron transport into mitochondria is an essential conserved process in eukaryotes, required for heme synthesis and FeS cluster assembly. Proposed studies will be performed in yeast mitochondria, but the organization of mitochondria is highly conserved between yeast and humans. The particular yeast proteins involved in iron transport and use in mitochondria have human orthologs (frataxin, mitochondrial carriers). Defects in these proteins have been implicated in porphyria, anemia and neurodegeneration, and so results obtained in with yeast will be relevant to human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK053953-17
Application #
8643784
Study Section
Special Emphasis Panel (ZRG1-HEME-C (02))
Program Officer
Bishop, Terry Rogers
Project Start
1998-07-01
Project End
2016-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
17
Fiscal Year
2014
Total Cost
$366,557
Indirect Cost
$137,459
Name
University of Pennsylvania
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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
Yoon, Heeyong; Knight, Simon A B; Pandey, Alok et al. (2015) Turning Saccharomyces cerevisiae into a Frataxin-Independent Organism. PLoS Genet 11:e1005135
Pandey, Alok; Pain, Jayashree; Ghosh, Arnab K et al. (2015) Fe-S cluster biogenesis in isolated mammalian mitochondria: coordinated use of persulfide sulfur and iron and requirements for GTP, NADH, and ATP. J Biol Chem 290:640-57
Yoon, Heeyong; Knight, Simon A B; Pandey, Alok et al. (2014) Frataxin-bypassing Isu1: characterization of the bypass activity in cells and mitochondria. Biochem J 459:71-81
Pandey, Alok; Gordon, Donna M; Pain, Jayashree et al. (2013) Frataxin directly stimulates mitochondrial cysteine desulfurase by exposing substrate-binding sites, and a mutant Fe-S cluster scaffold protein with frataxin-bypassing ability acts similarly. J Biol Chem 288:36773-86
Pandey, Alok; Yoon, Heeyong; Lyver, Elise R et al. (2012) Identification of a Nfs1p-bound persulfide intermediate in Fe-S cluster synthesis by intact mitochondria. Mitochondrion 12:539-49
Pandey, Alok; Golla, Ramesh; Yoon, Heeyong et al. (2012) Persulfide formation on mitochondrial cysteine desulfurase: enzyme activation by a eukaryote-specific interacting protein and Fe-S cluster synthesis. Biochem J 448:171-87
Yoon, Heeyong; Golla, Ramesh; Lesuisse, Emmanuel et al. (2012) Mutation in the Fe-S scaffold protein Isu bypasses frataxin deletion. Biochem J 441:473-80
Pandey, Alok; Yoon, Heeyong; Lyver, Elise R et al. (2011) Isd11p protein activates the mitochondrial cysteine desulfurase Nfs1p protein. J Biol Chem 286:38242-52

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