Inoue Kentaro, Keegstra Kenneth
MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA.
Plant J. 2003 Jun;34(5):661-9. doi: 10.1046/j.1365-313x.2003.01755.x.
Toc75 is a protein translocation channel in the outer envelope membrane of chloroplasts and its presence is essential for the biogenesis of the organelles. Toc75 is the only protein identified so far in the outer membrane of chloroplasts or mitochondria that is synthesized as a larger precursor, preToc75, with a bipartite transit peptide. Its N-terminus targets the protein to the stroma and is removed by the stromal processing peptidase, whereas its C-terminus mediates envelope targeting and is removed by a yet unknown peptidase. Several conserved domains have been identified in the C-terminal portion of the preToc75 transit peptide from six plant species. We evaluated their importance in the biogenesis of Toc75 by means of deletion or site-directed mutagenesis, followed by import experiments using isolated chlroplasts. Among the conserved domains, a polyglycine stretch was found to be necessary for envelope targeting. Substitution of this domain with other stretches of a single amino acid such as alanine caused mistargeting of the protein into the stroma, indicating an important role for this domain. Furthermore, a glutamate at +2 and two alanine residues at -3 and -1 to the second cleavage site were found to be important for processing. A potential mechanism for the biogenesis of Toc75 is discussed.
Toc75是叶绿体外被膜中的一种蛋白质转运通道，其存在对于细胞器的生物发生至关重要。Toc75是迄今为止在叶绿体或线粒体外膜中鉴定出的唯一一种作为较大前体preToc75合成的蛋白质，带有一个双功能转运肽。其N端将蛋白质靶向基质，并被基质加工肽酶去除，而其C端介导外被膜靶向，并被一种未知的肽酶去除。在来自六种植物物种的preToc75转运肽的C端部分鉴定出了几个保守结构域。我们通过缺失或定点诱变，然后使用分离的叶绿体进行导入实验，评估了它们在Toc75生物发生中的重要性。在保守结构域中，发现一段多聚甘氨酸序列对于外被膜靶向是必需的。用其他单氨基酸序列（如丙氨酸）替换该结构域会导致蛋白质错误靶向到基质中，表明该结构域具有重要作用。此外，发现第二个切割位点上游+2处的一个谷氨酸以及-3和-1处的两个丙氨酸残基对于加工很重要。文中还讨论了Toc75生物发生的潜在机制。
