Updated project metadata. Protein-protein interactions are fundamental to all cellular activities, with the ribosome increasingly recognized as a central platform steering the dynamics of nascent-chain interactions. In our focus on conserved N-terminal acetyltransferases (NATs), we identified distinct co-translational assembly pathways. This distinction persisted even among highly homologous subunits, with some playing contrasting roles. Notably, we found that only a few residues act as “hotspots”, initiating co-translational assembly interactions as they emerge from the ribosome exit tunnel. The significance of these hotspots was further validated in vivo through N-terminomics studies.