Maturation of Frataxin Within Mammalian and Yeast Mitochondria: One-Step Processing by Matrix Processing Peptidase

Donna M. Gordon 1 Qi Shi 1 Andrew Dancis 0 1 Debkumar Pain 1 0 Department of Medicine, University of Pennsylvania School of Medicine , Philadelphia, PA 19104-6100, USA 1 Department of Physiology, University of Pennsylvania School of Medicine , D403 Richards Building, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA Friedreich's ataxia is a neurodegenerative disease caused by mutations in the nuclear gene encoding frataxin (FRDA). FRDA is synthesized with an N-terminal signal sequence, which is removed after import into mitochondria. We have shown that FRDA was imported efficiently into isolated mammalian or yeast mitochondria. In both cases, the processing cleavage that removed the N-terminal signal sequence occurred in a single step on import, generating mature products of identical mobility. The processing cleavage could be reconstituted by incubating the FRDA preprotein with rat or yeast matrix processing peptidase (MPP) expressed in Escherichia coli. We used these assays to evaluate the import and processing of an altered form of FRDA containing the disease-causing I154F mutation. No effects on import or maturation of this mutated FRDA were observed. Likewise, no effects were observed on import and maturation of the yeast frataxin homolog (Yfh1p) carrying a homologous I130F mutation. These results argue against the possibility that the I154F mutation interferes with FRDA function via effects on maturation. Other mutations can be screened for effects on FRDA biogenesis as described here, by evaluating import into isolated mitochondria and by testing maturation with purified MPP. - Friedreichs ataxia (FA) is a neurodegenerative disease with an autosomal recessive pattern of inheritance. It is the most common inherited ataxia with a prevalence of 1 in 50 000 individuals (reviewed in ref. 1). The progressive neurodegeneration in FA primarily affects dorsal root ganglia in the central nervous system. The neurological symptoms include gait and limb ataxia, lower limb areflexia, loss of proprioception and dysarthria. Patients also develop skeletal abnormalities, hypertrophic cardiomyopathy and, often, impaired glucose tolerance (2). These symptoms progress with age, causing most patients to die before they reach the age of 30 years. The disease results from inherited defects in the gene that encodes a protein designated frataxin (FRDA). The most common molecular cause of FA is the hyperexpansion of a polymorphic GAA trinucleotide repeat (<39 repeat units in normal individual versus 661700 in FA patients) located in the first intron of the FRDA gene, resulting in a reduced level of FRDA mRNA (3,4). Several FA patients carry one allele with a hyperexpansion of the GAA repeat element and one allele with a missense mutation (46). The most common disease-causing missense mutation in FRDA is I154F. Another missense mutation in FRDA, G130V, in combination with a hyperexpanded allele, is associated with a milder and more slowly progressing disease course. The FA gene was identified by positional cloning (4). The encoded protein, FRDA, was localized to mitochondria by immunofluorescence and immunoelectron microscopy (3). Major clues about the cellular function of the protein came from work in the model organism Saccharomyces cerevisiae (712). Yeast possesses a homologous protein, Yfh1p (yeast frataxin homolog), which like its human counterpart is localized to yeast mitochondria (79). The deletion of the corresponding gene from the haploid organism results in respiratory deficiency because of the loss of mitochondrial DNA (8). Furthermore, D yfh1 yeast mutants accumulate iron within mitochondria (7,11). The yeast mutant phenotype is mirrored by the cells of affected tissues from patients with FA. Endomyocardial biopsies of FA patients show diminished activity of mitochondrial respiratory complexes (12) and high iron accumulation is found in heart tissue from patients with FA (13). Finally, FRDA and Yfh1p have highly homologous sequences at their C-termini and the human protein expressed in the yeast mutant is able to complement some of the mutant phenotypes if the first 39 amino acids of the FRDA precursor are replaced by the first 34 amino acids of Yfh1p. The I154F disease-associated mutation in the mature FRDA protein reduces its ability to complement the yeast mutant (8). Examination of the amino acid sequences of FRDA and its homologs does not immediately shed light on how the protein controls the build up of iron in organelles. We have previously proposed a link between Yfh1p maturation and its function in mitochondrial iron homeostasis (9). We demonstrated that on import into mitochondria the maturation of Yfh1p takes place in two steps: an initial proteolytic cleavage removing ~2 kDa and a subsequent cleavage removing an additional ~4 kDa from the N-terminus of the preprotein. The second step of processing is delayed in a yeast mutant lacking the mitochondrial chaperone Ssq1p. Furthermore, the phenotypes of ssq1-1 and D yfh1 yeast mutants are very similar (79,11,14 16). The most striking similarity is that both mutants exhibit an increase in mitochondrial iron content. Based on these results, the two-step processing of Yfh1p seems important in mitochondrial iron homeostasis in yeast (9). The possibility that FRDA exerts its effects via interactions with other mitochondrial proteins has been examined. Using a yeast two-hybrid assay, Koutnikova et al. (17) have identified the mitochondrial matrix processing peptidase (MPP) as an FRDA partner protein. These studies apparently suggest that the maturation of FRDA takes place in two steps: MPP cleavage of FRDA first results in an intermediate form that is processed further to the mature form. Furthermore, the diseasecausing I154F mutation appears to slow the processing of FRDA by MPP. The slower processing rate of the mutated protein is thought to contribute to a functional FRDA deficiency in FA patients (17). However, these processing experiments were done in vitro with bacterially expressed rat MPP and in vivo by overexpression of frataxin in COS cells followed by immunoblotting. A more definitive conclusion requires that import and maturation experiments be carried out using purified mitochondria. Such studies for FRDA are lacking. Here we describe the import and maturation of wild-type and I154F mutant FRDA using purified mammalian and yeast (wild-type and ssq1-1 mutant) mitochondria. Surprisingly, in all cases imported FRDA was processed to the mature form in one step by MPP. No intermediate form of FRDA was detected during its maturation within the organelle. In addition, we found that the I154F mutation had no detectable effect on import and/or maturation of FRDA. These results demonstrate that, although the C-terminal domains of FRDA and Yfh1p are functionally homologous, the processing of their N-terminal signal sequences within mitochondria is quite different. These studies provide the basis for understan (...truncated)