The tailored chaperone pathway for eukaryotic elongation factor 1A (eEF1A) biogenesis

Abstract

Eukaryotic translation elongation factor 1A (eEF1A) is a highly abundant, multi-domain GTPase essential to protein translation. Using a combination of yeast genetics, microscopy analysis, biochemical reconstitution, and structural modeling in S. cerevisiae, we find that eEF1A requires a dedicated chaperone pathway composed of three components for its productive and efficient folding. The absence of these factors in cells causes acute proteotoxicity driven by misfolded and depleted levels of eEF1A and the induction of cellular stress responses. We find that the essential Zinc-finger protein 1 (Zpr1) is absolutely required for eEF1A biogenesis, and using in vitro assays and structural modeling, we show that Zpr1 enables folding of eEF1A through contacts with its zinc-finger and alpha-helical hairpin domains, organizing eEF1A into its mature folded state. Zpr1 also highly stimulates the GTPase activity of eEF1A, in a sense “testing” its functionality prior to client release. Altered Inheritance of Mitochondria protein 29 (Aim29), though not essential, recognizes eEF1A in the GTP-bound pre-hydrolysis conformation and enables efficient recycling from Zpr1. Aim29 dampens the Zpr1•eEF1A GTPase activity and facilitates eEF1A’s exit from the folding cycle. The last non-essential factor Ypl225w works upstream of Zpr1 and Aim29 to co-translationally fold the first domain of eEF1A. 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). Ypl225w primes eEF1A to bind GTP, which upon binding stimulates rapid release of Ypl225w. Our work uncovers the role of these uncharacterized proteins as part of an ATP-independent chaperone pathway dedicated to the folding of nascent eEF1A.