Structural remodeling of ribosome associated Hsp40-Hsp70 chaperones during co-translational folding

Abstract

Ribosome associated complex (RAC), an obligate heterodimer of HSP40 and HSP70 (Zuo1 and Ssz1 in yeast), is conserved in eukaryotes and functions as co-chaperone for another HSP70 (Ssb1/2 in yeast) to facilitate co-translational folding of nascent polypeptides. Many mechanistic details, such as the coordination of one HSP40 with two HSP70s and the dynamic interplay between RAC-Ssb and growing nascent chains, remain unclear. Here, we report three sets of structures of RAC-containing ribosomal complexes isolated from Saccharomyces cerevisiae. Structural analyses indicate that RAC on the nascent-chain-free ribosome is in an autoinhibited conformation, and in the presence of a nascent chain at the peptide tunnel exit (PTE), RAC undergoes large-scale structural remodeling to make Zuo1 J-Domain more accessible to Ssb. Our data also suggest a role of Zuo1 in orienting Ssb-SBD proximal to the PTE for easy capture of the substrate. Altogether, in accordance with previous data, our work suggests a sequence of structural remodeling events for RAC-Ssb during co-translational folding, triggered by the binding and passage of growing nascent chain from one to another.

Chaperone requirements for de novo folding of Saccharomyces cerevisiae septins

“…Protein structures are AlphaFold predictions (the NTE of Cdc3 is hidden, for clarity) or cryo-EM structures (human CCT; PDB 7LUM [ Knowlton et al. , 2021 ]; Zuo1–Ssz1, PDB 7 × 3K [ Chen et al. , 2022 ]).…”

Volleying plasma membrane proteins from birth to death: Role of J-domain proteins

“…The mode of ER import depends on the amino acid composition of the polypeptide. Different Hsp70:JDP machineries assist these pathways to stabilize the translation elongation, prevent misfolding, aggregation, or facilitate interaction with the ER translocon complex ( Ast et al, 2013 ; Aviram and Schuldiner, 2017 ; Chen et al, 2022 ) ( Figure 2 ). During protein synthesis, the Ssb1/2 (yeast)/Hsp70L1 (mammals), together with Ribosome Associated Complex (RAC), functions to regulate the folding and targeting of nascent polypeptides to the ER.…”

“…RAC is a heterodimer composed of Hsp70, Ssz1 (HSPA14 in mammals), and JDP, Zuo1 (DNAJC2 in mammals) ( Hundley et al, 2005 ; Otto et al, 2005 ). Upon nascent chain elongation, Ssz1 and Zuo1 undergo a conformational change, thus making the J-domain of Zuo1 accessible to binding with another Hsp70, Ssb1/2 ( Chen et al, 2022 ). Ssb interacts with a large number of ribosome-associated nascent chains as well as the cytosolic factor, signal recognition particle (SRP), to facilitate efficient translation and ER targeting of proteins ( Pfund et al, 1998 ; Willmund et al, 2013 ; Döring et al, 2017 ; Shiber et al, 2018 ).…”

“…Ssb interacts with a large number of ribosome-associated nascent chains as well as the cytosolic factor, signal recognition particle (SRP), to facilitate efficient translation and ER targeting of proteins ( Pfund et al, 1998 ; Willmund et al, 2013 ; Döring et al, 2017 ; Shiber et al, 2018 ). Together, Ssb and Zuo1 help in co-translational protein folding and their targeting to the ER ( Kramer et al, 2019 ; Chen et al, 2022 ). Although deletion of Ssb leads to widespread misfolding and aggregation of newly synthesized polypeptides ( Koplin et al, 2010 ), its requirement in the biogenesis or maturation of membrane proteins is unknown.…”

“…In different organisms, specific Hsp70: JDPs mediate the ER targeting of PM proteins by (A) CTT or (B) PTT. During both CTT and PTT, ribosome-associated Hsp70:JDPs Y,H interact and protect the nascent polypeptides ( Otto et al, 2005 ; Döring et al, 2017 ; Chen et al, 2022 ). PTT occurs through multiple pathways (1) GET/TRC40 mdiated Y, (2) Chaperone mediated H or (3) alternate pathways N ( Rabu et al, 2009 ; Casson et al, 2017 ; Cho et al, 2021 ).…”