HYPK promotes N-terminal protein acetylation through rapid ribosome exchange of NatA

Numerous protein biogenesis factors cotranslationally facilitate the maturation of nascent proteins. Among them, N-terminal acetyltransferase A (NatA) acetylates the N terminus of ∼40% of the eukaryotic proteome. NatA is bound to Huntingtin-interacting protein K (HYPK), which inhibits NatA activity in vitro but enhances function in vivo. Here, kinetic and in-cell measurements resolve this paradox, showing that HYPK acts as a ribosome exchange factor for NatA. Without HYPK, hyper-tight ribosome binding prevents NatA from accessing additional ribosomes following each round of acetylation. HYPK accelerates NatA dissociation from the ribosome to license multiple turnovers, allowing a sub-stoichiometric level of this enzyme to globally acetylate the nascent proteome. Our results uncover a previously unidentified function of HYPK and demonstrate that a "Goldilocks" zone of ribosome interaction kinetics is required for cotranslational protein biogenesis machineries to act on all translating ribosomes in the cell.