A molecular switch in NAC prevents mitochondrial protein mistargeting by SRP

The nascent polypeptide-associated complex (NAC) co-translationally screens all nascent proteins and regulates their access to signal recognition particle (SRP) to ensure the fidelity of protein targeting to the endoplasmic reticulum (ER). However, the mechanism by which NAC prevents the mistargeting of nascent mitochondrial proteins remains unclear. Here, we identify a molecular switch in NAC that allows its central barrel domain to adopt a stabilized conformation on ribosomes exposing a mitochondrial targeting sequence (MTS). Mutations of the MTS on the nascent chain or in the NAC switch region increase NAC barrel dynamics and reduce its binding to the ribosome. This impairs the ability of NAC to prevent mistargeting by SRP and causes ER stress in human cells. Our work reveals how NAC detects nascent mitochondrial proteins early in translation and prevents their promiscuous access to SRP, elucidating the structural basis that underlies this role and providing mechanistic insights into protein targeting fidelity with broader implications for cellular proteostasis. How ribosome-bound NAC distinguishes mitochondrial precursors from ER clients has remained unclear. Here, authors reveal a molecular switch in NAC that limits SRP access to nascent mitochondrial precursors and prevents their mistargeting to the ER.