Membrane protein insertion through a mitochondrial β-barrel gate Caroline Lindau, Alexandra I.C. Höhr, Christophe Wirth, Jian Qiu, David A. Stroud, Stephan Kutik, Bernard Guiard*, Carola Hunte, Thomas Becker, Nikolaus Pfanner, Nils Wiedemann Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany; *Centre de Génétique Moléculaire, CNRS, 91190 Gif-sur-Yvette, France
The sorting and assembly machinery (SAM) and the β-barrel assembly machinery (BAM) are required for the sorting and assembly of β-barrel proteins in eukaryotic cells and bacteria. The essential homologous core subunits of these machineries, Sam50/BamA, belong to the Omp85 superfamily. Mitochondrial β-barrel precursors are synthesized in the cytosol, translocated across the outer mitochondrial membrane through the translocase of the outer membrane and guided by the small TIM chaperones through the intermembrane space to SAM. Finally, SAM recognizes the β-signal of the precursor and folds the precursor into the outer membrane. High-resolution structures of the Omp85 protein BamA show flexible interactions of the first and last β-strand and suggest a membrane thinning on this side. Different models are hypothesized for the insertion of β-barrel proteins. In the assisting model, precursors are inserted at the protein-lipid interface of Sam50/BamA. In the budding model, precursors are translocated through the Sam50/BamA channel and are then laterally released into the outer membrane by opening the Sam50/BamA barrel between the first and last β-strand. To analyze the mechanism of β-barrel membrane insertion, we performed in vitro import studies of β-barrel precursors into Sam50 variants containing single cysteine residues at defined positions. Upon cysteine specific crosslinking and disulfide bond scanning between precursor and Sam50, we were able to track the precursor during its import. Based on our data, imported β-barrel precursors are translocated through the Sam50 interior. The endogenous β-signal of Sam50 is replaced by the β-signal of the incoming precursor and folds between the first and last β-strand of Sam50. Subsequently further β-hairpins are folded at the lateral gate until the completely folded precursor is released into the outer membrane. Since the β-signal and the Omp85 core components are conserved, we speculate that this is a general mechanism of β-barrel protein biogenesis in mitochondria, chloroplasts and bacteria.
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