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