Biogenesis of mitochondrial outer membrane proteins involves their integration into the lipid bilayer. Among these proteins are those that form a single-span topology, but our understanding of their biogenesis is scarce. In this study, we found that the MIM complex is required for the membrane insertion of some single-span proteins. However, other such proteins integrate into the membrane in a MIM-independent manner. Moreover, the biogenesis of the studied proteins was dependent to a variable degree on the TOM receptors Tom20 and Tom70. We found that Atg32 C-terminal domain mediates dependency on Tom20, whereas the cytosolic domains of Atg32 and Gem1 facilitate MIM involvement. Collectively, our findings (1) enlarge the repertoire of MIM substrates to include also tail-anchored proteins, (2) provide new mechanistic insights to the functions of the MIM complex and TOM import receptors, and (3) demonstrate that the biogenesis of MOM single-span proteins shows variable dependence on import factors.

线粒体外膜蛋白的生物发生涉及它们整合到脂质双层中。这些蛋白质中有形成单跨拓扑的蛋白质，但我们对其生物发生的了解很少。在这项研究中，我们发现MIM复合物是某些单跨度蛋白膜插入所必需的。但是，其他此类蛋白质也以MIM独立的方式整合到膜中。此外，所研究蛋白质的生物发生在一定程度上取决于TOM受体Tom20和Tom70。我们发现Atg32 C末端域介导了对Tom20的依赖性，而Atg32和Gem1的胞质域促进了MIM的参与。总的来说，我们的发现（1）扩大了MIM底物的范围，使其还包括尾锚蛋白，