The mitochondrial outer membrane contains so-called β-barrel proteins, which allow communication between the cytosol and the mitochondrial interior^1,2,3. Insertion of β-barrel proteins into the outer membrane is mediated by the multisubunit mitochondrial sorting and assembly machinery (SAM, also known as TOB)^4,5,6. Here we use cryo-electron microscopy to determine the structures of two different forms of the yeast SAM complex at a resolution of 2.8–3.2 Å. The dimeric complex contains two copies of the β-barrel channel protein Sam50—Sam50a and Sam50b—with partially open lateral gates. The peripheral membrane proteins Sam35 and Sam37 cap the Sam50 channels from the cytosolic side, and are crucial for the structural and functional integrity of the dimeric complex. In the second complex, Sam50b is replaced by the β-barrel protein Mdm10. In cooperation with Sam50a, Sam37 recruits and traps Mdm10 by penetrating the interior of its laterally closed β-barrel from the cytosolic side. The substrate-loaded SAM complex contains one each of Sam50, Sam35 and Sam37, but neither Mdm10 nor a second Sam50, suggesting that Mdm10 and Sam50b function as placeholders for a β-barrel substrate released from Sam50a. Our proposed mechanism for dynamic switching of β-barrel subunits and substrate explains how entire precursor proteins can fold in association with the mitochondrial machinery for β-barrel assembly.

线粒体外膜含有所谓的 β 桶蛋白，它允许胞质溶胶和线粒体内部^1,2,3之间的通信。β-桶蛋白插入外膜是由多亚基线粒体分选和组装机制（SAM，也称为 TOB）介导的^4,5,6. 在这里，我们使用低温电子显微镜以 2.8-3.2 Å 的分辨率确定两种不同形式的酵母 SAM 复合物的结构。二聚体复合物包含两个拷贝的 β 桶通道蛋白 Sam50——Sam50a 和 Sam50b——具有部分打开的侧向门。外周膜蛋白 Sam35 和 Sam37 从胞质侧盖住 Sam50 通道，对二聚体复合物的结构和功能完整性至关重要。在第二个复合物中，Sam50b 被 β-桶蛋白 Mdm10 取代。与 Sam50a 合作，Sam37 通过从胞质侧穿透其横向封闭的 β 桶内部来招募和捕获 Mdm10。载有底物的 SAM 复合体包含 Sam50、Sam35 和 Sam37 各一个，但既不包含 Mdm10 也不包含第二个 Sam50，表明 Mdm10 和 Sam50b 作为从 Sam50a 释放的 β-桶底物的占位符。我们提出的动态转换 β-桶亚基和底物的机制解释了整个前体蛋白如何与用于 β-桶组装的线粒体机制相关地折叠。