Transmembrane Traffic of Proteins with Unnatural Amino Acids

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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