Membrane protein insertion through a mitochondrial β-barrel gate

Making your way through the side of a barrel The mechanism of membrane insertion and assembly of b-barrel proteins is a central question of outer membrane biogenesis of mitochondria, chloroplasts, and Gram-negative bacteria. Höhr et al. developed assays to address this fundamental problem. They systematically mapped precursor proteins transported by the mitochondrial Omp85 channel (Sam50) to elucidate the entire membrane insertion pathway of a precursor in the native membrane environment. Their findings directly demonstrate translocation of precursor proteins through the lumen of the mitochondrial Omp85 channel, signal recognition by β-strand exchange between channel and precursor, and exit through the lateral gate into the membrane. Science, this issue p. eaah6834 The mechanism by which mitochondrial outer membrane proteins are inserted is elucidated. INTRODUCTION The outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts characteristically contain β-barrel membrane proteins. These proteins contain multiple amphipathic β strands that form a closed barrel. This arrangement exposes hydrophobic amino acid residues to the lipid phase of the membrane, with polar residues facing the lumen of the barrel. β-barrel proteins form outer membrane channels for protein import and export, and for metabolite and nutrient exchange. An essential step in the biogenesis of β-barrel proteins is their insertion into the outer membrane. The β-barrel assembly machinery (BAM) of bacteria and the sorting and assembly machinery (SAM) of mitochondria are crucial for the membrane insertion of β-barrel precursors. The core subunits of these machineries, BamA and Sam50, are homologous 16-stranded β-barrel proteins that belong to the outer membrane protein family 85 (Omp85). The β signal located in the last β strand of the precursor initiates protein insertion into the outer membrane; however, the molecular mechanism of β-barrel insertion has not been understood. Controversial models about the role of BAM and SAM have been discussed. These models either favor precursor translocation into the BamA or Sam50 barrel followed by lateral release through an opened β-barrel gate or suggest membrane thinning and precursor insertion at the BamA or Sam50 protein-lipid interface. RATIONALE Structural studies have suggested that BamA and Sam50 harbor a dynamic lateral gate formed between β strands 1 and 16. In addition, BamA and Sam50 have been proposed to induce a thinning of the lipid bilayer near the lateral gate. To determine the translocation pathway during β-barrel membrane insertion, we probed the proximity of β-barrel precursors (Tom40, Por1, VDAC1) to Sam50 in intact mitochondria of the model organism baker’s yeast, Saccharomyces cerevisiae. We engineered precursors and Sam50 variants with cysteine residues at defined positions and mapped the environment of precursors in transit by disulfide-bond scanning and cysteine-specific cross-linking. RESULTS Our findings indicated that during transport of β-barrel precursors by the SAM complex, the lateral gate of Sam50 between β strands 1 and 16 was open and contained accumulated precursor. The β signal of the precursor specifically interacted with β strand 1 of Sam50 and thus replaced the endogenous β signal (β strand 16) of Sam50. Precursor transfer to the lateral gate occurred via the channel lumen of Sam50 and required the conserved loop 6 located in the channel. β hairpin–like elements consisting of two antiparallel β strands of the precursor were translocated and inserted into the lateral gate. The precursor remained associated with the Sam50 gate until the folded full-length β-barrel protein was released into the outer membrane. CONCLUSION Our findings indicate that β-barrel precursors are inserted into the lumen of the Sam50 channel and are released into the mitochondrial outer membrane via the opened lateral gate of Sam50. The carboxy-terminal β signal of the precursor initiates opening of the gate by exchange with the endogenous Sam50 β signal. An increasing number of β hairpin–like loops of the precursor accumulate at the lateral gate. Upon folding at Sam50, the full-length β-barrel protein is laterally released into the outer membrane. Membrane thinning in the vicinity of the lateral gate likely facilitates insertion of the protein into the lipid bilayer. Thus, the membrane-insertion pathway of β-barrel proteins combines elements of both controversially discussed models: transport through the lumen of Sam50 and the lateral gate and subsequent insertion into the thinned membrane next to the gate. Owing to the conservation of both the β signal and Omp85 core machinery, we speculate that β-signal exchange, folding at the gate, and lateral release into the membrane represent a general mechanism for β-barrel protein biogenesis in mitochondria, chloroplasts, and Gram-negative bacteria. β-Barrel protein insertion via the lateral gate of Sam50. β-Barrel precursors are transferred through the Sam50 interior to the lateral gate, which is formed by β strands 1 and 16. Upon gate opening, the β signal of the precursor substitutes for the endogenous Sam50 β signal. A conserved loop of Sam50 promotes β-signal binding to the gate and insertion of subsequent β hairpins. The folded β-barrel protein is released into the outer membrane. Po, polar amino acid residue; G, glycine; Hy, hydrophobic amino acid residue; C, C terminus; IRGF, binding motif. The biogenesis of mitochondria, chloroplasts, and Gram-negative bacteria requires the insertion of β-barrel proteins into the outer membranes. Homologous Omp85 proteins are essential for membrane insertion of β-barrel precursors. It is unknown if precursors are threaded through the Omp85-channel interior and exit laterally or if they are translocated into the membrane at the Omp85-lipid interface. We have mapped the interaction of a precursor in transit with the mitochondrial Omp85-channel Sam50 in the native membrane environment. The precursor is translocated into the channel interior, interacts with an internal loop, and inserts into the lateral gate by β-signal exchange. Transport through the Omp85-channel interior followed by release through the lateral gate into the lipid phase may represent a basic mechanism for membrane insertion of β-barrel proteins.