General protein folding is mediated by chaperones that utilize ATP hydrolysis to regulate client binding and release. Zinc-finger protein 1 (Zpr1) is an essential ATP-independent chaperone dedicated t Show more
General protein folding is mediated by chaperones that utilize ATP hydrolysis to regulate client binding and release. Zinc-finger protein 1 (Zpr1) is an essential ATP-independent chaperone dedicated to the biogenesis of eukaryotic translation elongation factor 1A (eEF1A), a highly abundant GTP-binding protein. How Zpr1-mediated folding is regulated to ensure rapid Zpr1 recycling remains an unanswered question. Here, we use yeast genetics and microscopy analysis, biochemical reconstitution, and structural modeling to reveal that folding of eEF1A by Zpr1 requires GTP hydrolysis. Furthermore, we identify the highly conserved altered inheritance of mitochondria 29 (Aim29) protein as a Zpr1 co-chaperone that recognizes eEF1A in the GTP-bound, pre-hydrolysis conformation. This interaction dampens Zpr1⋅eEF1A GTPase activity and facilitates client exit from the folding cycle. Our work reveals that a bespoke ATP-independent chaperone system has mechanistic similarity to ATPase chaperones but unexpectedly relies on client GTP hydrolysis to regulate the chaperone-client interaction. Show less
The conserved regulon of heat shock factor 1 in budding yeast contains chaperones for general protein folding as well as zinc-finger protein Zpr1, whose essential role in archaea and eukaryotes remain Show more
The conserved regulon of heat shock factor 1 in budding yeast contains chaperones for general protein folding as well as zinc-finger protein Zpr1, whose essential role in archaea and eukaryotes remains unknown. Here, we show that Zpr1 depletion causes acute proteotoxicity driven by biosynthesis of misfolded eukaryotic translation elongation factor 1A (eEF1A). Prolonged Zpr1 depletion leads to eEF1A insufficiency, thereby inducing the integrated stress response and inhibiting protein synthesis. Strikingly, we show by using two distinct biochemical reconstitution approaches that Zpr1 enables eEF1A to achieve a conformational state resistant to protease digestion. Lastly, we use a ColabFold model of the Zpr1-eEF1A complex to reveal a folding mechanism mediated by the Zpr1's zinc-finger and alpha-helical hairpin structures. Our work uncovers the long-sought-after function of Zpr1 as a bespoke chaperone tailored to the biogenesis of one of the most abundant proteins in the cell. Show less