What are the functional domains of frataxin?
Mutational analyses of yeast and human frataxin will be carried out in S. cerevisiae to identified amino acids required for assembly, iron storage, and iron release. Select mutants will be expressed in E. coli and analyzed along with the wild type protein in vitro.
How do frataxin monomers assemble into multimers?
Gel filtration, sedimentation equilibrium analysis, dynamic light scattering, and atomic force microscopy will be used to study the pathway of frataxin assembly.
What are the roles of dioxygen and iron-catalyzed radical reactions in frataxin assembly?
The initial steps of frataxin assembly - iron binding and oligomerization - will be studied under both aerobic and anaerobic conditions, and in the presence of radical generating systems or anti-oxidants.
How does iron loading of frataxin occur?
O2 consumption and H+ release measurements will be used to define whether frataxin has ferroxidase activity, and to study the iron oxidation and hydrolysis chemistry involved in iron loading.
What is the structure of the frataxin multimer and how does it enable frataxin to store iron?
We will provide recombinant protein to Dr. Rosenzweig, who will attempt crystallization and analysis of the three-dimensional structure of the frataxin multimer.
How is iron released from frataxin and what is the biologic importance of this release?
Chelators, reductants, and potential physiologic acceptor molecules will be used to study iron release from frataxin.
What are the size, shape, and iron content of frataxin multimers in vivo?
Native frataxin multimers will be isolated from yeast and their properties characterized.
Mitochondrial processing peptidases and iron homeostasis
We have shown that Mitochondrial Intermediate Peptidase (MIP) (http://www.ncbi.nlm.nih.gov/Omim 602241) cooperates functionally with frataxin to maintain mitochondrial iron homeostasis in S. cerevisiae (Branda et al. 1999a), and that a similar interaction may also exist in mammalian cells (Chew et al. 2000) (click on these references for free reprints). We have proposed that yeast frataxin maintains mitochondrial iron homeostasis (i) directly, by promoting iron export, and (ii) indirectly, by regulating MIP activity, which stimulates mitochondrial iron uptake. An important finding was that the yeast MIP (Oct1p) is induced in Yfh1p-depleted yeast, and exacerbates the mitochondrial iron accumulation that results from loss of Yfh1p. This implies that human MIP could be one of the as yet unknown factors that influence the clinical variability of FRDA. Different genetic variants of HMIP could be associated with constitutively different rates of mitochondrial iron uptake, which could change the degree of iron accumulation in FRDA. This hypothesis is supported by our findings that human and yeast MIP are functionally interchangeable in yeast, and that the MIP and frataxin mRNAs are similarly and predominantly expressed in tissues with high oxygen consumption, including heart and various regions of the brain. Therefore, we have genetically mapped the HMIP gene (MIPEP) and have defined its genomic structure (Chew et al. 2000).
Future directions & projects
This work has opened the way to molecular studies of MIPEP in FRDA, and possibly other degenerative diseases with iron imbalance. We are now developing a mouse line in which the frataxin gene carries a HygroB-poly(A) cassette in the first intron. In this way, most primary transcripts will be polyadenylated, and therefore end, at the strong poly(A) site in the cassette. Based on a study that used this approach, we should obtain mice with 15-30% residual frataxin expression, within the range observed in FRDA (6-30%). This line will enable us to study the effects of frataxin deficiency in mammalian cells. We are also developing a mutant mouse MIP line. By crossing FRDA and MIP mutant mice, we can test if MIP defects attenuate the effects of frataxin deficiency.
In analyzing whether MIP might be involved in the maturation of frataxin, we demonstrated that Yfh1p and human frataxin (fxn) are actually processed to the mature form in two sequential steps by another component of the import machinery, the Mitochondrial Processing Peptidase (MPP)
(http://www.ncbi.nlm.nih.gov/Omim 603131) (Branda et al. 1999b; Cavadini et al. 2000b). This pattern of processing sets frataxin apart from most mitochondrial proteins, typically cleaved to mature form in a single step by MPP. We have also shown that MPP cleaves the precursor and intermediate form of human frataxin at different rates, and that the second step limits the levels of mature protein produced within mitochondria. The targeting signals of Yfh1p and human frataxin are 51 and 55 residues long, 1.5-2 times the average mitochondrial presequence (20-40 residues). We have shown that they are cleaved between 20-21 and 51-52 (Yfh1p), and 41-42 and 55-56 (human frataxin).
Future directions & projects
Plant ferritins reside in the chloroplast and are synthesized as larger precursors that are also processed in two steps. Much like two-step processing of frataxin, the functional significance of two-step processing of plant ferritin is unknown. However, in the case of plant ferritin the first cleavage occurs upon import into the chloroplast, while the second takes place after ferritin assembly and has been observed during germination, in vivo, and during iron release, in vitro. We are therefore testing the possibility that two-step processing regulates assembly/iron storage by frataxin.