Identification and characterization of novel eukaryotic chaperones

Abstract

Protein folding and assembly is aided by molecular chaperones that prevent aggregation of unfolded or partially folded intermediates and guide them to their native folded state. Chaperones engage clients through cycles of binding and release from aggregation-prone, non-native regions. 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. 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. We identified the highly conserved altered inheritance of mitochondria 29 (Aim29) as an eEF1A biogenesis factor. Structural modeling using AlphaFold-Multimer suggested that Aim29 senses the GTP-bound conformation of eEF1A folding intermediates bound to Zpr1. We validated this prediction using yeast genetics, cell biological and biochemical reconstitution approaches, and uncovered that Aim29 sensing of GTP-bound eEF1A coupled to a GTP hydrolysis event facilitates eEF1A exit from the folding cycle and allows for Zpr1 recycling. 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. Next, we attempted to use AlphaFold-Multimer to identify additional chaperones or biogenesis factors that haven’t yet been uncovered. We optimized an Alphafold-based pipeline for screening a protein of interest against the entire yeast proteome to identify high-confidence interactors. The success of this screening approach is highlighted by two examples. First, we identified the previously uncharacterized but conserved eukaryotic protein Ypl225w as an eEF1A chaperone candidate and subsequent work by another graduate student in the lab revealed that Ypl225w was a ribosome-associating chaperone that mediates GTP-driven vectorial folding of nascent eEF1A. We also applied this pipeline to an essential eukaryotic GTPase of unknown function, Drosophila melanogaster Misato-Like protein (Dml1). The top interactors for Dml1 were subunits of the chaperonin-containing T-complex (CCT), which we validated experimentally. Acute depletion of Dml1 lead to a decrease in levels of assembled CCT and accumulation of monomers. This observation, along with structural modeling and other preliminary results suggest that Dml1 could be an assembly chaperone for CCT.