Schmidt G W, Bartlett S G, Grossman A R, Cashmore A R, Chua N H
J Cell Biol. 1981 Nov;91(2 Pt 1):468-78. doi: 10.1083/jcb.91.2.468.
We have used an in vitro reconstitution system, consisting of cell-free translation products and intact chloroplasts, to investigate the pathway from synthesis to assembly of two polypeptide subunits of the light-harvesting chlorophyll-protein complex. These polypeptides, designated 15 and 16, are integral components of the thylakoid membranes, but they are products of cytoplasmic protein synthesis. Double immunodiffusion experiments reveal that the two polypeptides share common antigenic determinants and therefore are structurally related. Nevertheless, they are synthesized in vitro from distinct mRNAs to yield separate precursors, p15 and p16, each of which is 4,000 to 5,000 daltons larger than its mature form. In contrast to the hydrophobic mature polypeptides, the precursors are soluble in aqueous solutions. Along with other cytoplasmically synthesized precursors, p15 and p16 are imported into purified intact chloroplasts by a post-translational mechanism. The imported precursors are processed to the mature membrane polypeptides which are recovered exclusively in the thylakoids. The newly imported polypeptides are assembled correctly in the thylakoid lipid bilayer and they bind chlorophylls. Thus, these soluble membrane polypeptide precursors must move from the cytoplasm through the two chloroplast envelope membranes, the stroma, and finally insert into the thylakoid membranes, where they assemble with chlorophyll to form the light-harvesting chlorophyll protein complex.
我们使用了一种体外重建系统，该系统由无细胞翻译产物和完整的叶绿体组成，以研究光捕获叶绿素 - 蛋白质复合体的两个多肽亚基从合成到组装的途径。这些多肽，命名为15和16，是类囊体膜的组成成分，但它们是细胞质蛋白质合成的产物。双向免疫扩散实验表明，这两种多肽具有共同的抗原决定簇，因此在结构上相关。然而，它们在体外由不同的mRNA合成，产生单独的前体，p15和p16，每个前体比其成熟形式大4000至5000道尔顿。与疏水的成熟多肽不同，前体可溶于水溶液。与其他细胞质合成的前体一起，p15和p16通过翻译后机制被导入纯化的完整叶绿体中。导入的前体被加工成成熟的膜多肽，这些多肽仅在类囊体中回收。新导入的多肽在类囊体脂质双层中正确组装，并与叶绿素结合。因此，这些可溶性膜多肽前体必须从细胞质穿过两层叶绿体包膜、基质，最终插入类囊体膜中，在那里它们与叶绿素组装形成光捕获叶绿素蛋白质复合体。
