Coupling of Mitochondrial Import and Export Translocases by Receptor-Mediated Supercomplex Formation

The mitochondrial outer membrane harbors two protein translocases that are essential for cell viability: the translocase of the outer mitochondrial membrane (TOM) and the sorting and assembly machinery (SAM). The precursors of β-barrel proteins use both translocases—TOM for import to the intermembrane space and SAM for export into the outer membrane. It is unknown if the translocases cooperate and where the β-barrel of newly imported proteins is formed. We established a position-specific assay for monitoring β-barrel formation in vivo and in organello and demonstrated that the β-barrel was formed and membrane inserted while the precursor was bound to SAM. β-barrel formation was inhibited by SAM mutants and, unexpectedly, by mutants of the central import receptor, Tom22. We show that the cytosolic domain of Tom22 links TOM and SAM into a supercomplex, facilitating precursor transfer on the intermembrane space side. Our study reveals receptor-mediated coupling of import and export translocases as a means of precursor channeling. [Display omitted] [Display omitted] •Native assay monitors β-barrel folding of imported proteins in intact mitochondria•β-barrel folding and membrane insertion occur at sorting and assembly machinery SAM•Receptor Tom22 connects mitochondrial import and export translocases TOM and SAM•Translocase supercomplex promotes precursor channeling The import and export translocases TOM and SAM collaborate, forming a supercomplex that promotes the channeling of precursor proteins from one translocase to the other during the assembly of β-barrel proteins in the mitochondrial membrane.