Biochem. J. (2010) 427, 313–321 (Printed in Great Britain) doi:10.1042/BJ20100015 313 A novel EP-involved pathway for iron release from soya bean seed ferritin Xiaoping FU*, Jianjun DENG*, Haixia YANG*, Taro MASUDA†, Fumiyuki GOTO‡, Toshihiro YOSHIHARA‡ and Guanghua ZHAO* 1 *CAU and ACC Joint Laboratory of Space Food, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China, †Laboratory of Food Quality Design and Development, Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan, and ‡Biotechnology Sector, Environmental Science Research Laboratory, Central Research Institute of Electric Power Industry, 1646 Abiko, Chiba 270-1194, Japan Iron in phytoferritin from legume seeds is required for seedling germination and early growth. However, the mechanism by which phytoferritin regulates its iron complement to these physiological processes remains unknown. In the present study, protein degrad- ation is found to occur in purified SSF (soya bean seed ferritin) (consisting of H-1 and H-2 subunits) during storage, consistent with previous results that such degradation also occurs during seedling germination. In contrast, no degradation is observed with animal ferritin under identical conditions, suggesting that SSF autodegradation might be due to the EP (extension peptide) on the exterior surface of the protein, a specific domain found only in phytoferritin. Indeed, EP-deleted SSF becomes stable, confirming the above hypothesis. Further support comes from a protease activity assay showing that EP-1 (corresponding to the EP of the H-1 subunit) exhibits significant serine protease-like activity, whereas the activity of EP-2 (corresponding to the EP of the H-2 subunit) is much weaker. Consistent with the observation above, rH-1 (recombinant H-1 ferritin) is prone to degradation, whereas its analogue, rH-2, becomes very stable under identical conditions. This demonstrates that SSF degradation mainly originates from the serine protease-like activity of EP-1. Associated with EP degradation is a considerable increase in the rate of iron release from SSF induced by ascorbate in the amyloplast (pH range, 5.8– 6.1). Thus phytoferritin may have facilitated the evolution of the specific domain to control its iron complement in response to cell iron need in the seedling stage. Key words: autodegradation, extension peptide, iron release, phytoferritin, serine protease-like activity. INTRODUCTION Ferritins are a class of multimeric iron storage and detoxification proteins. The importance of their dual function is underscored by their ubiquitous distribution throughout all organisms, with the exception of fungi. The ferritin complex has 24 subunits assembled into a spherical shell characterized by a 432-point symmetry. Up to 4500 Fe 3+ atoms can be stored either as the crystalline mineral ferrihydrite or as amorphous hydrous ferric oxyphosphate in the inner cavity of the assembled ferritin shell [1–3]. Structural analyses of vertebrate, plant and bacterial ferritins indicate that each subunit consists of a four-helix bundle (helices A, B, C and D) and a fifth short helix (helix E) [1,4,5]. In mammals, two distinct ferritin subunits (H and L) are found with similar three-dimensional structures. The H subunit has ferroxidase centres responsible for fast Fe 2+ oxidation. In contrast, L subunits lack ferroxidase centres, and thus do not exhibit fast Fe 2+ oxidation kinetics, but facilitate nucleation of the mineral core [5]. Amino acids involved in the definition of the ferroxidase centre are strictly conserved in all plant ferritins except for PSF (pea seed ferritin), where a histidine residue is replaced with a glutamic acid residue at position 62 of the amino acid sequence in the ferroxidase centre [6,7]. However, plant and animal ferritins are remarkably different in their cytological localization. In contrast with animal ferritin existing in the cell cytoplasm, plants store iron within ferritin mainly in plastids, such as the amyloplast in seeds [3]. Unlike animal ferritin, in which two types of subunits (H and L) occur, only the H-type subunit has been described in phytoferritin. Ferritin from dried soya bean seed consists of two subunits, a 26.5 kDa (H-1) subunit and a 28.0 kDa (H-2) subunit, which share ∼ 80 % amino acid sequence identity [8,9]. The two subunits are encoded by two distinct genes, SferH-1 (GenBank ® accession number M64337) and SferH-2 (GenBank ® accession number AB062754) respectively [8], and are synthesized as a precursor (32 kDa) that contains a TP (transit peptide) and a following EP (extension peptide) at its N-terminal. The TP is responsible for the precursor targeting plastids [10]. Upon transport to the plastids, the TP is cleaved from the subunit precursor, producing the mature subunit which assembles in a 24-mer ferritin within the plastids [11]. Thus in mature phytoferritin, 24 EP domains per molecule represent the major structural difference between animal and plant ferritin. In PSF, each EP domain is composed of 24 amino acid residues, 11 of which form a specific α-helix, termed the P-helix, flanked by proline residues (X and L) [6]. Differing from the TP in function, it was recently discovered that the EP serves as a second binding and ferroxidase centre contributing to iron core mineralization at high ferritin iron loadings (> 48 iron/ protein shell) [12], indicative of the role of the EP in iron oxidative deposition in phytoferritin. Although Arabidopsis ferritin is considered crucial to protecting cells against oxidative damage rather than for iron storage [13], in legume seeds, the majority of total iron is stored in ferritin in the amyloplast; such storage is known to meet plant demand during seedling germination and growth [1,14]. Therefore elucidating whether or not the EP plays a role in phytoferritin iron release in the seedling stage is the focus of the present study. In the present study, protein degradation was found to occur upon SSF (soya bean seed ferritin) standing at 4 ◦ C, an observation consistent with a previous report where PSF showed the same Abbreviations used: AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; AMC, 7-amino-4-methylcoumarin; Boc, t-butoxycarbonyl; EP, extension peptide; MALDI–TOF-MS, matrix-assisted laser-desorption ionization–time-of-flight MS; MCA, (7-methoxycoumarin-4-yl)acetyl; PSF, pea seed ferritin; rH-1 (rH-2), recombinant H-1 (H-2) ferritin; SSF, soya bean seed ferritin; TP, transit peptide; WT, wild-type. 1 To whom correspondence should be addressed (email gzhao1000@yahoo.com). c  The Authors Journal compilation c  2010 Biochemical Society www.biochemj.org Biochemical Journal