Protein Association and Dissociation Regulated by Extension Peptide: A Mode for Iron Control by Phytoferritin in Seeds

Abstract

Most of the iron in legume seeds is stored in ferritin located in the amyloplast, which is used during seed germination. However, there is a lack of information on the regulation of iron by phytoferritin. In this study, soluble and insoluble forms of pea (Pisum sativum) seed ferritin (PSF) isolated from dried seeds were found to be identical 24-mer ferritins comprising H-1 and H-2 subunits. The insoluble form is favored at low pH, whereas the two forms reversibly interconvert in the pH range of 6.0 to 7.8, with an apparent pK a of 6.7. This phenomenon was not observed in animal ferritins, indicating that PSF is unique. The pH of the amyloplast was found to be approximately 6.0, thus facilitating PSF association, which is consistent with the role of PSF in long-term iron storage. Similar to previous studies, the results of this work showed that protein degradation occurs in purified PSF during storage, thus proving that phytoferritin also undergoes degradation during seedling germination. In contrast, no degradation was observed in animal ferritins, suggesting that this degradation of phytoferritin may be due to the extension peptide (EP), a specific domain found only in phytoferritin. Indeed, removal of EP from PSF significantly increased protein stability and prevented degradation under identical conditions while promoting protein dissociation. Correlated with such dissociation was a considerable increase in the rate of ascorbate-induced iron release from PSF at pH 6.0. Thus, phytoferritin may have facilitated the evolution of EP to enable it to regulate iron for storage or complement in seeds.

Overexpression of wheat ferritin gene TaFER-5B enhances tolerance to heat stress and other abiotic stresses associated with the ROS scavenging

“…In sea lettuce ferritins, the extension peptide contributes to shell stability and surface hydrophobicity [29]. The removal of the extension peptide in pea seed ferritin both increases protein stability and promotes the reversible dissociation of the mature ferritin protein [30]. The secondary structure of pea seed ferritin is highly similar to that of mammalian ferritin [31].…”

Isolation and characterization of a new phytoferritin from broad bean (Vicia faba) seed with higher stability compared to pea seed ferritin

“…However, such protein degradation was not observed in animal ferritins such as HoSF (horse spleen ferritin) and HuHF (human H-chain ferritin) under the same experimental conditions. Interestingly, no protein degradation occurs upon removal of the EP from PSF and SSF (Fu, Deng, Yang, Masuda, Goto, Yoshihara, & Zhao, 2010;Yang, Fu, Li, Leng, Chen, & Zhao, 2010). These findings demonstrated that phytoferritin degradation comes from the EP, a specific domain only located in plant ferritin, but not from any contaminants such as various proteases.…”

“…Additionally, similar results were obtained with other buffers, such as Mops or Mes in the 6.0-7.5 pH range (results not shown), and protein samples from three different protein preparations/purifications, suggesting that the observed degradation is not sample/buffer/pH dependent. In contrast to BBSF, PSF began to degrade at the 5th day to a great extent, and the degree of its degradation is much larger as compared to that of BBSF (Yang, Fu, Li, Leng, Chen, & Zhao, 2010). Similar to PSF, SSF is also unstable and the degree of its protein degradation is likewise much stronger than that of BBSF.…”

“…The protein was incubated with SDS-PAGE sample buffer (containing 25% glycerol, 12.5% 0.5 mol/ L Tris-HCl, pH 6.8, 2% SDS, 1% bromophenol blue and 5% β-mercaptoethanol) at 100°C for 5 min at different time intervals. Then the mixtures were incubated at −20°C to be used for SDS-PAGE (Yang, Fu, Li, Leng, Chen, & Zhao, 2010).…”

“…However, recent studies have shown that known plant ferritins such as soybean seed ferritin (SSF) and pea seed ferritin (PSF) are unstable because a ratio of H-1 to H-2 is higher, usually ranging from 1:2 to 1:1 Yang et al, 2010). Thus, it is important to find a new ferritin with a lower amount of the H-1.…”