NAC promotes co-translational protein folding at the ribosomal tunnel exit

The nascent polypeptide-associated complex (NAC) coordinates enzymatic modifications and membrane targeting of nascent chains during translation. While the role of NAC as a dynamic hub for other factors is well established, its direct role in co-translational folding is unclear. By proteome-wide profiling of co-translational NAC 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 in mitochondrial membrane protein biogenesis. Together, these findings show that human NAC acts as a bona fide co-translational chaperone that directly promotes early protein folding at the ribosomal tunnel exit, expanding its functional repertoire in protein biogenesis. • Selective ribosome profiling maps NAC’s co-translational nascent chain interactome • The NAC β-barrel domain dynamically engages nascent chains and the ribosome surface • NAC assists early folding steps of cytonuclear protein domains at the tunnel exit • NAC uses a tunnel-exit-oriented hydrophobic patch to bind and fold nascent chains Santos et al. reveal NAC’s co-translational interactome, showing compartment-specific nascent chain interactions in human cells. NAC engages emerging chains at the ribosomal tunnel exit via a ribosome-oriented hydrophobic pocket, promoting early domain folding without stabilizing intermediate structures. These findings establish NAC as a co-translational chaperone integrating folding with protein biogenesis.