Most plant seeds contain 11S globulins as major storage proteins for their nutrition. Soybean glycinin belongs to the 11S globulin family and consists of five kinds of subunits. We determined the crystal structure of a homohexamer of the glycinin A3B4 subunit at 2.1-Å resolution. The crystal structure shows that the hexamer has 32-point group symmetry formed by face-to-face stacking of two trimers. The interface buries the highly conserved interchain disulfide. Based on the structure, we propose that an ingenious face-to-face mechanism controls the hexamer formation of the 11S globulin by movement of a mobile disordered region to the side of the trimer after posttranslational processing. Electrostatic analysis of the faces suggests that the interchain disulfide-containing face has high positive potential at acidic pH, which induces dissociation of the hexamer into trimers that may be susceptible to proteinases after seed imbibition. This dissociation might result in the degradation and mobilization of 11S globulins as storage proteins in embryos during germination and seedling growth. P lants accumulate protein reserves in developing seeds to act as a sink of nitrogen, sulfur, and carbon. Most dicotyledonous plant seeds contain 7S and͞or 11S globulins and albumins as the major storage proteins in the embryo or cotyledons, whereas some cereals such as wheat, barley, and corn contain prolamins in the endosperm (1, 2). The 11S globulins are distributed more widely than the 7S globulins among plant seeds. Although the quaternary structures of the 7S and 11S globulins are different from each other (the former being a trimeric protein and the latter a hexameric protein), they are believed to be derived from a common ancestor because of the partial homologies in their amino acid sequences and limited proteolysis patterns (3). This assumption has been confirmed by x-ray crystallography of 7S globulins from kidney bean (4), jack bean (5), and soybean (6) and of the trimeric soybean 11S globulin precursor (7).In general, 11S globulins are composed of several kinds of subunits. For example, five major subunits have been identified from soybean: A1aB1b, A2B1a, A1bB2, A3B4, and A5A4B3. In developing seeds, the constituent subunits of 11S globulin are synthesized as a single polypeptide precursor, preproprotein, the signal sequence of which is removed cotranslationally. The resultant proproteins assemble into trimers of Ϸ8S in the endoplasmic reticulum. The proprotein trimers are transported from the endoplasmic reticulum to protein storage vacuoles (PSVs), where they then are cleaved to form acidic and basic polypeptides that are linked by a disulfide bond (8). Finally, the mature proteins assemble into hexamers. The protein reserves are stored in the dormant seed until its germination.Recently we determined the crystal structure of the soybean proglycinin A1aB1b homotrimer at 2.8-Å resolution (7). The protomer consists of N-and C-terminal modules that each include a jelly-roll ␤-barrel and a helix domain. However, the packa...

Background: In eukaryotes, chromosomal DNA is licensed to be replicated through the sequential loading of the origin recognition complex, Cdc6 and mini-chromosome maintenance protein complex (MCM) onto chromatin. However, how the replication machinery is assembled onto the licensed chromatin during initiation of replication is poorly understood.

Ferritins are important iron storage and detoxification proteins that are widely distributed in living kingdoms. Because plant ferritin possesses both a ferroxidase site and a ferrihydrite nucleation site, it is a suitable model for studying the mechanism of iron storage in ferritin. This article presents for the first time the crystal structure of a plant ferritin from soybean at 1.8-Å resolution. The soybean ferritin 4 (SFER4) had a high structural similarity to vertebrate ferritin, except for the N-terminal extension region, the C-terminal short helix E, and the end of the BC-loop. Similar to the crystal structures of other ferritins, metal binding sites were observed in the iron entry channel, ferroxidase center, and nucleation site of SFER4. In addition to these conventional sites, a novel metal binding site was discovered intermediate between the iron entry channel and the ferroxidase site. This site was coordinated by the acidic side chain of Glu 173 and carbonyl oxygen of Thr 168 , which correspond, respectively, to Glu 140 and Thr 135 of human H chain ferritin according to their sequences. A comparison of the ferroxidase activities of the native and the E173A mutant of SFER4 clearly showed a delay in the iron oxidation rate of the mutant. This indicated that the glutamate residue functions as a transit site of iron from the 3-fold entry channel to the ferroxidase site, which may be universal among ferritins.

Ferritin is a multimeric iron storage protein composed of 24 subunits. Ferritin purified from dried soybean seed resolves into two peptides of 26.5 and 28 kDa. To date, the 26.5-kDa subunit has been supposed to be generated from the 28-kDa subunit by cleavage of the N-terminal region. We performed amino acid sequence analysis of the 28-kDa subunit and found that it had a different sequence from the 26.5-kDa subunit, thus rendering it novel among known soybean ferritins. We cloned a cDNA encoding this novel subunit from 10-dayold seedlings, each of which contained developed bifoliates, an epicotyl and a terminal bud. The 26.5-kDa subunit was found to be identical to that identified previously lacking the C-terminal 16 residues that correspond to the E helix of mammalian ferritin. However, the corresponding region in the 28-kDa soybean ferritin subunit identified in this study was not susceptible to cleavage. We present evidence that the two different ferritin subunits in soybean dry seeds show differential sensitivity to protease digestions and that the novel, uncleaved 28-kDa ferritin subunit appears to stabilize the ferritin shell by co-existing with the cleaved 26.5-kDa subunit. These data demonstrate that soybean ferritin is composed of at least two different subunits, which have cooperative functional roles in soybean seeds.

In eukaryotes, chromosomal DNA is licensed for a single round of replication in each cell cycle. Xenopus MCM3 protein has been implicated in the licensing of replication in egg extract. We have cloned cDNAs encoding five immunologically distinct proteins associated with Xenopus MCM3 as members of the MCM/P1 family. Six Xenopus MCM proteins formed a physical complex in the egg extract, bound to unreplicated chromatin before the formation of nuclei, and apparently displaced from replicated chromatin. The requirement of six XMCM proteins for the replication activity of the egg extract before nuclear formation suggests that their re‐association with replicated chromatin at the end of the mitotic cell cycle is a key step for the licensing of replication.

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Ferritins are ubiquitous iron storage proteins. Recently, we identified a novel metal-binding site, transit site, in the crystal structure of phytoferritin. To elucidate the function of the transit site in ferritin from other species, we prepared transit-site-deficient mutants of human H ferritin, E140A and E140Q, and their iron oxidation kinetics was analyzed. The initial velocities of iron oxidization were reduced in the variants, especially in E140Q. The crystal structure of E140Q showed that the side chain of the mutated Gln140 was fixed by a hydrogen bond, whereas that of native Glu140 was flexible. These results suggest that the conserved transit site also has a function to assist with the metal ion sequestration to the ferroxidase site in ferritins from vertebrates.

DNA polymerase (Pol) is thought to be involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the requirement of other replicative DNA polymerases, DNA polymerases ␣ and ␦ (Pol␣ and ␦), for chromosomal DNA replication has been well documented by genetic and biochemical studies, the precise role, if any, of Pol in chromosomal DNA replication is still obscure. Here we show, with the use of a cell-free replication system with Xenopus egg extracts, that Xenopus Pol is indeed required for chromosomal DNA replication. In Poldepleted extracts, the elongation step of chromosomal DNA replication is markedly impaired, resulting in significant reduction of the overall DNA synthesis as well as accumulation of small replication intermediates. Moreover, despite the decreased DNA synthesis, excess amounts of Pol␣ are loaded onto the chromatin template in Pol-depleted extracts, indicative of the failure of proper assembly of DNA synthesis machinery at the fork. These findings strongly suggest that Pol, along with Pol␣ and Pol␦, is necessary for coordinated chromosomal DNA replication in eukaryotic cells.