Characterization of the targeting signal in mitochondrial β-barrel proteins

Mitochondrial beta-barrel proteins are synthesized on cytosolic ribosomes and must be specifically targeted to the organelle before their integration into the mitochondrial outer membrane. The signal that assures such precise targeting and its recognition by the organelle remained obscure. In the present study we show that a specialized beta-hairpin motif is this long searched for signal. We demonstrate that a synthetic beta-hairpin peptide competes with the import of mitochondrial beta-barrel proteins and that proteins harbouring a beta-hairpin peptide fused to passenger domains are targeted to mitochondria. Furthermore, a beta-hairpin motif from mitochondrial proteins targets chloroplast beta-barrel proteins to mitochondria. The mitochondrial targeting depends on the hydrophobicity of the beta-hairpin motif. Finally, this motif interacts with the mitochondrial import receptor Tom20. Collectively, we reveal that beta-barrel proteins are targeted to mitochondria by a dedicated beta-hairpin element, and this motif is recognized at the organelle surface by the outer membrane translocase.