Transmembrane Traffic of Proteins with Unnatural Amino Acids

大多数线粒体蛋白质在胞质溶胶中作为前体蛋白合成，并且随后在线粒体中是重要的。前体蛋白跨线粒体外膜和内膜的转运需要两种膜中转运机制的协调功能。为了更详细地分析易位机制的组成部分与易位前体蛋白之间的相互作用，我们在pSu 9（1-69）-DHFR中位点特异性地掺入了一个带有光反应性交联基团的非天然氨基酸pBzPhe，并将其作为模型前体蛋白。通过体外诱变，构建了6个在多肽的特定位置沿着携带琥珀终止密码子的突变基因。在抑制性tRNA存在下，合成突变型pSu 9（1-69）-DHFR融合蛋白（T4、A17、A29、T44、A56和Y67），所述抑制性tRNA装载有pBz-Phe，并且通过c ...更多信息 用兔网织红细胞裂解物偶联转录/翻译。除T44外，所有蛋白质都被有效地合成。此外，含有pBz-Phe的突变融合蛋白可以有效地进入线粒体，由于跨膜转运需要前体蛋白的解折叠，因此甲氨蝶呤稳定融合蛋白DHFR结构域的三级结构可以阻断其进入线粒体，形成转运中间体。这些通过转运通道转运的转运中间体应该与转运机制的组分密切接触。因此，中间体的照射将导致融合蛋白与易位机制的组分交联。实际上，我们可以观察到依赖于引入的pBz-Phe的位置的交联产物。这些形式可能代表融合蛋白和易位机制的组分之间的交联产物，这是由于它们的特异性相互作用。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