The C-terminal GGAP motif of Hsp70 mediates substrate recognition and stress response in yeast

Abstract

The allosteric coupling of the highly conserved nucleotide- and substrate-binding domains of Hsp70 has been studied intensively. In contrast, the role of the disordered, highly variable C-terminal region of Hsp70 remains unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD motif binds to the tetratricopeptide-repeat domains of Hsp70 co-chaperones. Here, we discovered that the TVEEVD sequence of cytoplasmic Hsp70 (Ssa1) functions as a SUMO-interacting motif. A second C-terminal motif of ∼15 amino acids between the α-helical lid and the extreme C terminus, previously identified in bacterial and eukaryotic organellar Hsp70s, is known to enhance chaperone function by transiently interacting with folding clients. Using structural analysis, interaction studies, fibril formation assays, and functional assays, we investigated the individual contributions of the α-helical bundle and the C-terminal disordered region of Ssa1 in the inhibition of fibril formation of the prion protein Ure2. Our results revealed that although the α-helical bundle of the Ssa1 substrate-binding domain (SBDα) does not directly bind to Ure2, the SBDα enhances the ability of Hsp70 to inhibit fibril formation. We found that a 20-residue C-terminal motif in Ssa1, containing GGAP and GGAP-like tetrapeptide repeats, can directly bind to Ure2, the Hsp40 co-chaperone Ydj1, and α-synuclein, but not to the SUMO-like protein SMT3 or BSA. Deletion or substitution of the Ssa1 GGAP motif impaired yeast cell tolerance to temperature and cell-wall damage stress. This study highlights that the C-terminal GGAP motif of Hsp70 is important for substrate recognition and mediation of the heat shock response.

Comparative Characterization of Plasmodium falciparum Hsp70-1 Relative to E. coli DnaK Reveals the Functional Specificity of the Parasite Chaperone

“…The chimeric protein, KPf, exhibited the highest ATPase activity (both basal and PfHsp40 stimulated). While Hsp40 primarily binds to the NBD domain to stimulate ATP hydrolysis by Hsp70, it is also known to make contact with the C-terminus of Hsp70 [ 42 ]. It is thus possible that the contact that PfHsp40 makes with the C-terminus of the SBD of PfHsp70-1 (also present in KPf) is unique compared to that of DnaK.…”

Rapid deacetylation of yeast Hsp70 mediates the cellular response to heat stress

“…As described in our recent work, a C-terminal truncation of Ssa1 ΔGGAP displays a temperature sensitive ( ts ) phenotype related Hsp104 redundancy, which we consider a compensatory mechanism to adapt to heat shock 34 . To clarify whether the temperature sensitivity of the 4Q mutant is connected to Hsp104 function, we assessed the ability of WT, 4Q and 4R cells to grow on media containing 3 mM Gdn-HCl (an inhibitor of Hsp104 40 ).…”

Comparative characterisation ofPlasmodium falciparumHsp70-1 relative toE. coliDnaK reveals functional specificity of the parasite chaperone

“…The chimeric protein, KPf, exhibited the highest ATPase activity (both basal and PfHsp40 stimulated). While Hsp40 primarily binds to the NBD domain to stimulate ATP hydrolysis by Hsp70, it is also known to make contact with the C-terminus of Hsp70 (38). It is thus possible that the contact that PfHsp40 makes with the C-terminus of the SBD of PfHsp70-1 (also present in KPf) is unique compared to that of DnaK.…”