Molecular Cell Biology of Plants, Department of Biosciences, Center for Membrane Proteomics and Cluster of Excellence Macromolecular Complexes, Goethe University Frankfurt, Max von Laue Strasse 9/N200/R3.02, Frankfurt, Germany.
J Mol Biol. 2010 Sep 24;402(3):510-23. doi: 10.1016/j.jmb.2010.07.052. Epub 2010 Aug 3.
Protein translocation of cytosolically synthesized proteins requires signals for both targeting of precursor proteins to the surface of the respective compartment and their transfer across its membrane. In contrast to signals for peroxisomal and endoplasmic reticulum translocation, the signals for mitochondrial and chloroplast transport are less well defined with respect to length and amino acid requirements. To study the properties of signals required for translocation into chloroplasts in vitro and in vivo, we used fusion proteins composed of transit peptides and the Ig-like module of the muscle protein titin as passenger. We observed that about 60 amino acids-longer than the transit peptide length of many experimentally confirmed chloroplast proteins-are required for efficient translocation. However, within native chloroplast precursor proteins with transit peptides shorter than 60 amino acids, extension appears to be present as they are efficiently imported into organelles. In addition, the interaction of an unfolded polypeptide stretch of 60 or more amino acids with receptors at the chloroplast surface results in the unidirectionality of protein translocation into chloroplasts even in the presence of a competing C-terminal peroxisomal targeting signal. These findings prove the existing ideas that initial targeting is defined by the N-terminal signal and that the C-terminal signal is sensed only subsequently.
细胞质中合成的蛋白质的蛋白转位需要靶向前体蛋白到各自隔室表面的信号以及它们穿过其膜的转移的信号。与过氧化物酶体和内质网转位的信号相比，线粒体和叶绿体转运的信号在长度和氨基酸需求方面定义得不太明确。为了研究体外和体内进入叶绿体所需的信号的特性，我们使用融合蛋白作为载体，融合蛋白由转运肽和肌球蛋白的 Ig 样模块组成。我们观察到，对于有效的转运，需要比许多经过实验证实的叶绿体蛋白的转运肽长度长约 60 个氨基酸。然而，在天然的叶绿体前体蛋白中，转运肽的长度短于 60 个氨基酸，似乎存在延伸，因为它们被有效地导入细胞器。此外，与叶绿体表面的受体相互作用的 60 个或更多氨基酸的未折叠多肽延伸导致蛋白质单向进入叶绿体，即使存在竞争的 C 末端过氧化物酶体靶向信号也是如此。这些发现证明了现有的观点，即初始靶向由 N 末端信号定义，并且 C 末端信号仅随后被感知。
