The expression of human mitochondrial ferritin rescues respiratory function in frataxin-deficient yeast

Alessandro Campanella 2 Grazia Isaya 1 Heather A. O'Neill 1 Paolo Santambrogio 2 Anna Cozzi 2 Paolo Arosio 0 Sonia Levi 2 0 Dipartimento Materno Infantile e Tecnologie Biomediche, University of Brescia , 25125 Italy 1 Department of Pediatric and Adolescent Medicine, Mayo Clinic College of Medicine , Rochester, MN, USA 2 Department of Biological and Technological Research , IRCCS H. San Raffaele, Via Olgettina 58, Milano, 20132 Italy Mitochondrial ferritin (MtF) is structurally and functionally similar to the cytosolic ferritins, molecules designed to store and detoxify cellular iron. MtF expression in human and mouse is restricted to the testis and few tissues, and it is abundant in the erythroblasts of patients with sideroblastic anemia, where it is thought to protect the mitochondria from the damage caused by iron loading. Mitochondria iron overload occurs also in cells deficient in frataxin, a mitochondrial protein involved in iron handling and implicated in Friedreich ataxia. We expressed human MtF in frataxin-deficient yeast cells, a well-characterized model of mitochondrial iron overload and oxidative damage. The human MtF precursor was efficiently imported by yeast mitochondria and processed to functional ferritin that actively sequestered iron in the organelle. MtF expression rescued the respiratory deficiency caused by the loss of frataxin protecting the activity of iron - sulfur enzymes and enabling frataxin-deficient cells to grow on non-fermentable carbon sources. Furthermore, MtF expression prevented the development of mitochondrial iron overload, preserved mitochondrial DNA integrity and increased cell resistance to H2O2. The data show that MtF can substitute for most frataxin functions in yeast, suggesting that frataxin is directly involved in mitochondrial iron-binding and detoxification. - INTRODUCTION Iron is needed for the synthesis of enzymes essential for respiration, redox reactions and DNA synthesis, and is also potentially toxic for its capacity to catalyze free radical formation (1). Consequently iron homeostasis must be tightly controlled by specific mechanisms. The ones so far characterized are located at the cytoplasmic level and involve the iron regulatory proteins that sense iron levels, and the ferritins (2). These are 24-mer proteins composed of the H- and L-subunit types that sequester excess iron in their large cavity in a bioavailable and non-toxic form (1,3). However, much iron has to enter the mitochondria to be incorporated into heme and Fe/S complexes for the synthesis of enzymes (4,5), but little is known about the regulation of this trafficking and how iron toxicity is prevented. Mitochondrial ferritin (MtF) and frataxin are candidates to play important roles in the regulation of mitochondrial iron homeostasis, although their functional roles have not been fully elucidated. MtF has been recently identified in humans, primates and rodents, and shown to be encoded by an intronless gene, which in human is localized on chromosome 5q23.1 (6,7). It is expressed as a precursor with a long N-terminal extension (approximately 60 residues) that directs mitochondrial targeting. The amino acid sequence of the mature protein fully overlaps that of H-ferritin with 77% identity, including all the key residues for ferroxidase activity, and the study of the human and mouse recombinant proteins confirmed that they have iron-binding capacity and ferroxidase activity comparable to that of the well-characterized cytosolic H-ferritin (7). In addition, its crystallographic structure is remarkably similar to that of the H-ferritin (8). When the MtF precursor was expressed in HeLa cells it was found to be fully processed to the mature 21 kDa protein and to accumulate specifically inside the mitochondria. There it assembled in functional 24-mer ferritin molecules, which were active in taking up iron. This activity, which was linked to the integrity of the ferroxidase center, had a profound effect on cellular iron homeostasis, since it reduced both cytosolic ferritin levels and iron availability (9). It was concluded that MtF has a function similar to that of the wellcharacterized cytosolic ferritin, differing for the mitochondrial localization and for being composed of a single subunit type. MtFs lack the regulatory IRE sequence, and their expression does not seem to be iron-regulated at the post-transcriptional level (6). In addition, and at variance with the ubiquitous cytosolic ferritins, MtF is expressed in a limited number of tissues, mainly in the testis and spermatocytes (10). Interestingly, high levels of MtF protein have been found in ringed sideroblasts of patients with sideroblastic anemia (11). The mitochondria of these cells contain large iron deposits. Mutations in the ALAS2 gene are responsible for the genetic X-linked form of the disorder (12), whereas unknown factors, possibly linked to mitochondrial defects, are implicated in the sporadic forms (13). The excess iron is sequestered inside the MtF, and, because the sideroblasts live and proliferate, it has been suggested that MtF protects mitochondria from the toxicity of local iron excess (11). This hypothesis is supported by preliminary analysis of transfectant cells (10). Normal erythroblasts do not express detectable MtF, indicating that the protein is induced in the disorder. Frataxin deficiency is associated with Friedreich ataxia (FRDA), the most common genetic form of ataxia (reviewed in 14,15). It is a mitochondrial protein found in all eukaryotes including yeast. The mature protein is a monomer of 14 kDa, and its 3D structure does not show evident metal binding sites (16,17). However, it binds iron in vitro (18 21). It was shown that the yeast frataxin, Yfh1p, is activated by Fe(II) in the presence of O2 to form trimers that catalyze iron oxidation (22). Higher iron concentrations induce a stepwise assembly of the protein to higher oligomers that can sequester more than 2000 Fe atoms in ferrihydrite-like polynuclear structures, similar to those found in ferritins (23). In addition, Yfh1p oligomers can bind Fe(II) which is available to ferrochelatase for heme synthesis (21). In fact, a physical interaction between Yfh1p and ferrochelatase has been demonstrated in Biacore experiments (24). Other reports indicated that human frataxin can bind six to seven iron atoms that are donated to Isu1p, in the early stages of Fe/S cluster assembly (18). A physical interaction between Yfh1p and Isu1p in yeast has been demonstrated (25,26). Therefore, it has been proposed that Yfh1p has ferroxidase activity and iron storage properties which may protect the mitochondria from iron toxicity, and that it also acts as a chaperone to donate iron to the proteins involved in the two major pathways of iron utilization, Fe/S cluster assembly and heme synthesis. The relative importance of these two functions cannot be easily inferred by the effects of Yfh1p deficiency. In fact, yeast cells lac (...truncated)