Schnell D J, Blobel G
Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York 10021.
J Cell Biol. 1993 Jan;120(1):103-15. doi: 10.1083/jcb.120.1.103.
We have used a hybrid precursor protein to study the pathway of protein import into chloroplasts. This hybrid (pS/protA) consists of the precursor to the small subunit of Rubisco (pS) fused to the IgG binding domains of staphylococcal protein A. The pS/protA is efficiently imported into isolated chloroplasts and is processed to its mature form (S/protA). In addition to the mature stromal form, two intermediates in the pathway of pS/protA import were identified at early time points in the import reaction. The first intermediate represents unprocessed pS/protA bound to the outer surface of the chloroplast envelope and is analogous to a previously characterized form of pS that is specifically bound to the chloroplast surface and can be subsequently translocated in the stroma (Cline, K., M. Werner-Washburne, T. H. Lubben, and K. Keegstra. 1985. J. Biol. Chem. 260:3691-3696.) The second intermediate represents a partially translocated form of the precursor that remains associated with the envelope membrane. This form is processed to mature S/protA, but remains susceptible to exogenously added protease in intact chloroplasts. We conclude that the envelope associated S/protA is spanning both the outer and inner chloroplast membranes en route to the stroma. Biochemical and immunochemical localization of the two translocation intermediates indicates that both forms are exposed at the surface of the outer membrane at sites where the outer and inner membrane are closely apposed. These contact zones appear to be organized in a reticular network on the outer envelope. We propose a model for protein import into chloroplasts that has as its central features two distinct protein conducting channels in the outer and inner envelope membranes, each gated open by a distinct subdomain of the pS signal sequence.
我们使用了一种杂交前体蛋白来研究蛋白质导入叶绿体的途径。这种杂交体（pS/蛋白A）由核酮糖-1,5-二磷酸羧化酶小亚基的前体（pS）与葡萄球菌蛋白A的IgG结合结构域融合而成。pS/蛋白A能有效地导入分离的叶绿体，并被加工成其成熟形式（S/蛋白A）。除了成熟的基质形式外，在导入反应的早期时间点还鉴定出了pS/蛋白A导入途径中的两种中间体。第一种中间体代表未加工的pS/蛋白A结合在叶绿体被膜的外表面，类似于先前表征的一种pS形式，它特异性地结合在叶绿体表面，随后可在基质中转运（克莱恩，K.，M. 维尔纳-沃什伯恩，T. H. 卢布本，和K. 基斯特拉。1985年。《生物化学杂志》260:3691 - 3696）。第二种中间体代表前体的部分转运形式，它仍然与被膜膜结合。这种形式被加工成成熟的S/蛋白A，但在完整的叶绿体中仍易受外源添加的蛋白酶作用。我们得出结论，与被膜相关的S/蛋白A在进入基质的途中跨越了叶绿体的外膜和内膜。两种转运中间体的生化和免疫化学定位表明，这两种形式都在外膜表面与外膜和内膜紧密贴合的部位暴露。这些接触区域似乎在外被膜上组织成一个网状网络。我们提出了一个蛋白质导入叶绿体的模型，其核心特征是在外膜和内膜中有两个不同的蛋白质传导通道，每个通道由pS信号序列的一个不同亚结构域门控打开。
