The nascent polypeptide-associated complex (NAC) functions as a bona fide co-translational chaperone that recognizes emerging hydrophobic, α-helical segments within folded domains at the ribosomal tunnel exit via its β-barrel domain, directly promoting on-pathway protein folding while exhibiting compartment-specific roles in coordinating ER targeting and mitochondrial membrane protein biogenesis.

The nascent polypeptide-associated complex (NAC) coordinates enzymatic modifications and membrane targeting of nascent chains during translation. While NAC’s function as a dynamic hub for other factors is well-established, its direct role in co-translational folding is unclear. By proteome-wide profiling NAC co-translational interactions in human cells, we found that NAC recognizes emerging segments enriched in hydrophobicity and α-helical propensity, within folded domains of cytonuclear proteins. Single-molecule and structural analyses reveal that NAC, via its β-barrel domain, dynamically interacts with nascent chains at the ribosomal tunnel exit and is capable of promoting on-pathway folding. Compartment-specific nascent chain interactions of NAC further elucidate its role in targeting to the endoplasmic reticulum and mitochondrial membrane protein biogenesis. Together, these findings show that NAC acts as a bona fide co-translational chaperone that facilitates early protein folding at the ribosomal tunnel exit, expanding its functional repertoire in protein biogenesis.