The nascent polypeptide-associated complex (NAC) functions as a multifaceted cotranslational regulator that coordinates protein biogenesis through three distinct mechanisms: it recognizes hydrophobic and amphipathic helical motifs to chaperone domain folding in cytosolic, nuclear, ER, and mitochondrial proteins with compartment-specific timing; it uniquely senses nascent chains inside the ribosome exit tunnel through sequence-specific interactions that directly modulate translation elongation kinetics and control the early elongation ramp; and it selectively enhances organelle targeting by protecting transmembrane domains in mitochondrial precursors and coordinating with SRP for ER-destined proteins while preventing mistargeting of cytosolic proteins.

The nascent polypeptide-associated complex (NAC) is a conserved ribosome-bound factor with essential yet incompletely understood roles in protein biogenesis. Here, we show that NAC is a multifaceted regulator that coordinates translation elongation, cotranslational folding, and organelle targeting through distinct interactions with nascent polypeptides both inside and outside the ribosome exit tunnel. Using NAC-selective ribosome profiling in C. elegans, we identify thousands of sequence-specific NAC binding events across the nascent proteome, revealing broad cotranslational engagement with hydrophobic and helical motifs in cytosolic, nuclear, ER, and mitochondrial proteins. Unexpectedly, we discover an intra-tunnel sensing mode, where NAC engages ribosomes with extremely short nascent polypeptides inside the exit tunnel in a sequence-specific manner. These early NAC interactions induce an early elongation slowdown that tunes ribosome flux and prevent ribosome collisions, linking NAC’s chaperone activity to kinetic control of translation. We propose that NAC action protects aggregation-prone intermediates by shielding amphipathic helices thus promoting cytonuclear folding and supporting mitochondrial membrane protein biogenesis and ER targeting by early recognition of signal sequences and transmembrane domain. Our findings establish NAC as an early-acting, multifaceted orchestrator of cotranslational proteostasis, with distinct mechanisms of action on nascent chains depending on their sequence features and subcellular destinations.