Onda Yayoi, Kumamaru Toshihiro, Kawagoe Yasushi
Division of Plant Sciences, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan.
Proc Natl Acad Sci U S A. 2009 Aug 18;106(33):14156-61. doi: 10.1073/pnas.0904429106. Epub 2009 Aug 6.
The developing endosperm of rice (Oryza sativa, Os) synthesizes a large amount of storage proteins on the rough (r)ER. The major storage proteins, glutelins and prolamins, contain either intra or intermolecular disulfide bonds, and oxidative protein folding is necessary for the sorting of the proteins to the protein bodies. Here, we investigated an electron transfer pathway for the formation of protein disulfide bonds in the rER of the rice endosperm, focusing on the roles of the thiol-disulfide oxidoreductase, OsEro1. Confocal microscopic analysis revealed that N-glycosylated OsEro1 is localized to the rER membrane in the subaleurone cells, and that targeting of OsEro1 to the rER membrane depends on the N-terminal region from Met-1 to Ser-55. The RNAi knockdown of OsERO1 inhibited the formation of native disulfide bonds in the glutelin precursors (proglutelins) and promoted aggregation of the proglutelins through nonnative intermolecular disulfide bonds in the rER. Inhibition of the formation of native disulfide bonds was also observed in the seeds of the esp2 mutant, which lacks protein disulfide isomerase-like (PDIL)1;1, but shows enhanced OsEro1 expression. We detected the generation of H(2)O(2) in the rER of the WT subaleurone cells, whereas the rER-derived H(2)O(2) levels decreased markedly in EM49 homozygous mutant seeds, which have fewer sulfhydryl groups than the WT seeds. Together, we propose that the formation of native disulfide bonds in proglutelins depends on an electron transfer pathway involving OsEro1 and OsPDIL.
水稻（Oryza sativa，Os）发育中的胚乳在糙面内质网（rER）上合成大量贮藏蛋白。主要的贮藏蛋白，即谷蛋白和醇溶蛋白，含有分子内或分子间二硫键，氧化蛋白折叠对于这些蛋白分选到蛋白体中是必需的。在此，我们研究了水稻胚乳rER中蛋白二硫键形成的电子传递途径，重点关注硫醇-二硫氧化还原酶OsEro1的作用。共聚焦显微镜分析显示，N-糖基化的OsEro1定位于糊粉层下细胞的rER膜上，并且OsEro1靶向rER膜取决于从Met-1到Ser-55的N端区域。OsERO1的RNA干扰敲低抑制了谷蛋白前体（谷蛋白原）中天然二硫键的形成，并通过rER中非天然分子间二硫键促进了谷蛋白原的聚集。在esp2突变体种子中也观察到天然二硫键形成的抑制，该突变体缺乏类蛋白二硫键异构酶（PDIL）1;1，但OsEro1表达增强。我们检测到野生型糊粉层下细胞的rER中有H₂O₂生成，而在EM49纯合突变体种子中，rER衍生的H₂O₂水平显著降低，该突变体的巯基比野生型种子少。总之，我们提出谷蛋白原中天然二硫键的形成取决于涉及OsEro1和OsPDIL的电子传递途径。
