Principles of cotranslational mitochondrial protein import revealed by selective ribosome profiling

The biogenesis of nearly all mitochondrial proteins begins with translation on cytosolic ribosomes. How these proteins are subsequently delivered to mitochondria remains poorly understood. Here, we systematically investigated the coupling of mitochondrial protein translation and import using selective ribosome profiling in human cells. Cotranslational targeting requires an N-terminal presequence on the nascent protein and contributes to mRNA localization at the mitochondrial surface. This pathway is predominantly used by large, multidomain and topologically complex proteins, whose import efficiency is enhanced when targeted cotranslationally. In contrast to protein targeting to the endoplasmic reticulum (ER), cotranslational mitochondrial import does not favor membrane proteins and initiates late during translation, specifically upon the exposure of a complex globular fold in the nascent protein. Our findings reveal a multi-layered protein sorting system that recognizes both the targeting signal and protein folding status during translation. Overall design: Selective ribosome profiling of TOM22 in HEK293 cells to investigate cotranslational mitochondrial protein import.