Transmembrane Traffic of Proteins with Unnatural Amino Acids

大多数线粒体蛋白在细胞质中作为前体蛋白合成，随后对线粒体起重要作用。前体蛋白在线粒体内外膜上的易位需要两层膜上易位机制的协调作用。为了详细分析易位机制组成部分和易位前体蛋白之间的相互作用，我们特异性地加入了一种具有光反应性交联基团的非天然氨基酸，pBzPhe。我们使用由F_0-ATPase亚基9的序列组成的pSu9 (1-69)-DHFR作为融合到小鼠DHFR的模型前体蛋白。通过体外诱变，构建了6个在多肽特定位置携带琥珀色终止密码子的突变基因。突变体pSu9 (1-69)-DHFR融合蛋白（T4, A17, A29, T44， A56和Y67）在tRNA抑制下合成，tRNA与pBz-Phe和ri标记的Met通过c…More与兔网织细胞裂解液偶联转录/翻译。除T44外，其余蛋白均能高效合成。此外，含有pBz-Phe的突变融合蛋白可以有效地导入线粒体。由于跨线粒体膜的易位需要展开前体蛋白，甲氨蝶呤稳定融合蛋白的DHFR结构域的三级结构，阻止它们进入线粒体，导致易位中间体的形成。这些通过易位通道转运的易位中间体应该与易位机制的组成部分密切接触。因此，辐照中间体将导致融合蛋白与易位机制的组分交联。实际上，我们可以观察到交联产物依赖于引入的pBz-Phe的位置。这些形式可能代表了融合蛋白和易位机制组分之间的交联产物，这是由于它们之间的特定相互作用。少

Most mitochondrial proteins are synthesized as precursor proteins in the cytosol and are subsequently important into mitochondria. Translocation of precursor proteins across the mitochondrial outer and inner membranes requires coordinated functions of the translocation machineries in the two membranes. In order to analyze the interactions between components of the translocation machineries and translocating precursor proteins in details, we site-specifically incorporated an unnatural amino acid with a photoreactive cross-linking group, pBzPhe.We used pSu9 (1-69)-DHFR consisting of the presequence of the F_0-ATPase subunit 9 fused to mouse DHFR as a model precursor protein. Six mutant genes carrying the amber stop codon at specific positions along the polypeptide were constructed by in vitro mutagenesis. The mutant pSu9 (1-69)-DHFR fusion proteins (T4, A17, A29, T44, A56, and Y67) were synthesized in the presence of suppressor tRNA,which was charged with pBz-Phe, and RI-labeled Met by c … More oupled transcription/translation with rabbit reticulocyte lysate. All the proteins except for T44 were synthesized efficiently. Besides, mutant fusion proteins containing pBz-Phe could be imported into mitochondria efficiently.Since translocation across the mitochondrial membranes require unfolding of the precursor protein, stabilization of the tertiary structure of the DHFR domain of the fusion proteins by methotrexate blocks their import into mitochondria, resulting in the formation of translocation intermediates. These translocation intermediates in transit through the translocation channels should be in close contact with the components of the translocation machineries. Therefore irradiation of the intermediates will lead to cross-linking of the fusion proteins with the components of the translocation machineries. Indeed, we could observe cross-linked products which are dependent on the positions of introduced pBz-Phe. These forms may represent cross-linked products between the fusion proteins and the components of the translocation machineries due to their specific interactions. Less