Lee Kwang Hee, Kim Soo Jin, Lee Yong Jik, Jin Jing Bo, Hwang Inhwan
Center for Plant Intracellular Trafficking and Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea.
J Biol Chem. 2003 Sep 19;278(38):36794-805. doi: 10.1074/jbc.M304457200. Epub 2003 Jul 9.
Toc159, a protein located in the outer envelope membrane and the cytosol, is an important component of the receptor complex for nuclear-encoded chloroplast proteins. We investigated the molecular mechanism of protein import into chloroplasts by atToc159 using the ppi2 mutant, which has a T-DNA insertion at atToc159, shows an albino phenotype, and does not survive beyond the seedling stage due to a defect in protein import into chloroplasts. First we established that transiently expressing atToc159 in protoplasts obtained from the white leaf tissues of ppi2 plants complements the protein import defect into chloroplasts. Using this transient expression approach and a series of deletion mutants, we demonstrated that the C-terminal membrane-anchored (M) domain is targeted to the chloroplast envelope membrane in ppi2 protoplasts, and is sufficient to complement the defect in protein import. The middle GTPase (G) domain plays an additional critical role in protein import: the atToc159[S/N] and atToc159[D/L] mutants, which have a mutation at the first and second GTP-binding motifs, respectively, do not support protein import into chloroplasts. Leaf cells of transgenic plants expressing the M domain in a ppi2 background contained nearly fully developed chloroplasts with respect to size and density of thylakoid membranes, and displayed about half as much chlorophyll as wild-type cells. In transgenic plants, the isolated M domain localized to the envelope membrane of chloroplasts but not the cytosol. Based on these results, we propose that the M domain is the minimal structure required to support protein import into chloroplasts, while the G domain plays a regulatory role.
Toc159是一种位于外被膜和细胞质中的蛋白质，是核编码叶绿体蛋白受体复合物的重要组成部分。我们利用ppi2突变体研究了atToc159介导蛋白质导入叶绿体的分子机制。ppi2突变体在atToc159处有一个T-DNA插入，表现出白化表型，并且由于蛋白质导入叶绿体存在缺陷，在幼苗期后无法存活。首先，我们证实从ppi2植物的白色叶片组织获得的原生质体中瞬时表达atToc159可弥补蛋白质导入叶绿体的缺陷。利用这种瞬时表达方法和一系列缺失突变体，我们证明C末端膜锚定（M）结构域在ppi2原生质体中靶向叶绿体被膜，并且足以弥补蛋白质导入缺陷。中间的GTPase（G）结构域在蛋白质导入中发挥额外的关键作用：分别在第一个和第二个GTP结合基序处发生突变的atToc159[S/N]和atToc159[D/L]突变体不支持蛋白质导入叶绿体。在ppi2背景下表达M结构域的转基因植物的叶细胞，就类囊体膜的大小和密度而言，含有几乎完全发育的叶绿体，并且叶绿素含量约为野生型细胞的一半。在转基因植物中，分离的M结构域定位于叶绿体的被膜而不是细胞质。基于这些结果，我们提出M结构域是支持蛋白质导入叶绿体所需的最小结构，而G结构域起调节作用。
