A dedicated chaperone system essential for biogenesis of eukaryotic translation elongation factor 1A

Abstract

Protein folding is aided by molecular chaperones that prevent aggregation of unfolded or partially folded intermediates and guide them to their native states. Chaperones engage clients through cycles of binding and release from aggregation-prone non-native regions. Released clients can fold into native states or be recaptured for another chaperone cycle. ATP-dependent chaperones utilize ATP hydrolysis to drive chaperone conformational changes between low and high affinity states, while ATP-independent chaperones are regulated by diverse mechanisms. Expression of the core chaperone network is controlled by the transcriptional regulator Heat shock factor 1 (Hsf1). The Hsf1 regulon contains canonical chaperone machinery as well as the conserved and essential zinc-finger protein Zpr1, whose essential function in archaea and eukaryotes has remained unknown. We found that Zpr1 is a novel ATP-independent chaperone required for biogenesis of highly abundant eukaryotic translation elongation factor 1A (eEF1A). Zpr1 depleted cells experience severe protein folding stress driven by aggregation of newly synthesized eEF1A. Prolonged Zpr1 depletion causes eEF1A insufficiency, leading to translational stress and activation of the integrated stress response. Biochemical reconstitution of eEF1A folding showed that Zpr1 guides eEF1A intermediates to a protease-resistant state and indicated that GTP binding by eEF1A plays a role in this process. Further analysis demonstrated that Zpr1 chaperones eEF1A into a GTP hydrolysis-competent state, and perturbing GTP binding or hydrolysis severely inhibits the rate of eEF1A folding. Taken together, these results suggest a central role for eEF1A GTP hydrolysis in the Zpr1 mechanism. Next, we found that the uncharacterized protein Aim29 is an eEF1A biogenesis factor whose loss causes eEF1A aggregation and accompanying protein folding stress. Structural modeling using ColabFold suggesed that Aim29 senses the GTP-bound conformation of eEF1A folding intermediates bound to Zpr1. We validated this prediction using cell biological and biochemical reconstitution approaches and additionally found that Aim29 sensing of GTP-bound eEF1A coupled to a GTP hydrolysis event stimulates the release of eEF1A from the Zpr1 folding pathway. Our work identified a novel ATP-independent chaperone mechanism wherein the chaperone, Zpr1, and its co-chaperone, Aim29, enable a nucleotide hydrolysis event by their client to facilitate disassembly of the chaperone-client complex.