In mitochondria, β-barrel outer membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold β-barrel proteins and insert them into the mitochondrial outer membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transmembrane β-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the intermembrane space. Sam35 and Sam37 are located on the cytosolic side of the outer membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 β-barrel opens a lateral gate to accommodate its substrates.

β-barrel proteins are folded and inserted into outer membranes by multi-subunit protein complexes that are conserved across different types of outer membranes. In Gram-negative bacteria this complex is the b arrel- a ssembly m achinery (BAM), in mitochondria it is the s orting and a ssembly m achinery (SAM) complex, and in chloroplasts it is the o uter e nvelope p rotein Oep80. Mitochondrial β-barrel precursor proteins are translocated from the cytoplasm to the intermembrane space by the translocase of the o uter m embrane (TOM) complex, and stabilized by molecular chaperones before interaction with the assembly machinery. Outer membrane bacterial BamA interacts with four periplasmic accessory proteins, whereas mitochondrial Sam50 interacts with two cytoplasmic accessory proteins. Despite these major architectural differences between BAM and SAM complexes, their core proteins, BamA and Sam50, seem to function the same way. Based on the new SAM complex structures, we propose that the mitochondrial β-barrel folding mechanism follows the budding model with barrel-switching aiding in the release of new barrels. We also built a new molecular model for Tom22 interacting with Sam37 to identify regions that could mediate TOM-SAM supercomplex formation.

In mitochondria, b-barrel outer membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold b-barrel proteins and insert them into the mitochondrial outer membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transmembrane b-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the intermembrane space. Sam35 and Sam37 are located on the cytosolic side of the outer membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 bbarrel opens a lateral gate to accommodate its substrates. The SAM complex structure suggests how it interacts with other mitochondrial outer membrane proteins to create supercomplexes. components: a b-barrel core, Sam50, which spans the outer membrane, and two accessory subunits, Sam35 and Sam37, that associate with Sam50 on the cytosolic side of the membrane 2-5 . Sam50 and Sam35 are essential proteins, with Sam50 folding and inserting b-barrel substrates into the outer membrane and Sam35 interacting with the substrate b-signal located in the last b-strand 3 . Sam37, while not essential, functions in substrate release 3,6 and may also promote formation of a TOM-SAM supercomplex 7 .Bacterial b-barrel membrane proteins take a different pathway to reach the outer membrane, but they are inserted by evolutionarily related machinery 8 . In bacteria, proteins are synthesized in the cytoplasm, secreted across the inner membrane by the Sec translocon, bound to chaperones in the periplasm, and transferred to the Bacterial Assembly Machinery (BAM) complex for folding and insertion into the outer membrane 9,10 . In Escherichia coli, four lipoproteins, BamB, BamC, BamD, and BamE, associate with the periplasmic domain of BamA (itself a 16-stranded transmembrane b-barrel) to fold and insert b-barrels ranging in size and complexity from 8 b-strands with a simple barrel fold, to 26 b-strands with multiple domains 11 . Structures of BamA and BAM complexes illustrate two features of BamA that facilitate folding and insertion: [1] BamA has a narrowed hydrophobic surface where the first and last b-strands meet, which locally compresses and destabilizes the lipid bilayer to allow membrane protein insertion (BamA assisted model). [2] The first and last b-strands form a 'lateral gate' that has been shown to open and close by molecular dynamics (MD) simulations, disulfide crosslinking, and structures solved by X-ray crystallography and cryo-electron microscopy (cryoEM) 12-19 . The required opening and closing of the lateral gate gave rise to the budding model, where a b-