A ribosome-associating chaperone mediates GTP-driven vectorial folding of nascent eEF1A

Eukaryotic translation elongation factor 1A (eEF1A) is a highly abundant, multi-domain GTPase. Post-translational steps essential for eEF1A biogenesis are carried out by bespoke chaperones but co-translational mechanisms tailored to eEF1A folding remain unexplored. Here, we find that the N-terminal, GTP-binding domain of eEF1A is prone to co-translational misfolding and using computational approaches, yeast genetics, and microscopy analysis, we identify the conserved yet uncharacterized yeast protein Ypl225w as a chaperone dedicated to solving this problem. Proteomics and biochemical reconstitution reveal that Ypl225w’s interaction with ribosomal eEF1A nascent chains depends on additional binding of Ypl225w to the UBA domain of nascent polypeptide-associated complex (NAC). Lastly, we show by orthogonal chemical genetics that Ypl225w primes eEF1A nascent chains for their subsequent binding to GTP and release from Ypl225w. Our work establishes eEF1A as a model system for chaperone-dependent co-translational folding and unveils a novel mechanism for GTP-driven folding on the ribosome.