Comprehensive identification of HSP70/HSC70 Chaperone Clients in Human Cells

Abstract

The HSP70 family of chaperones are the front-line of protection from stress-induced misfolding and aggregation of polypeptides in most organisms and are responsible for promoting the stability, folding, and degradation of clients to maintain cellular protein homeostasis. Here we demonstrate quantitative identification of HSP70 and HSC70 clients using an ubiquitin-mediated proximity tagging strategy and show that, despite their high degree of similarity, these enzymes have largely non-overlapping specificities. Both proteins show a preference for association with newly synthesized polypeptides but each responds differently to changes in the stoichiometry of proteins in obligate multi-subunit complexes. In addition, expression of an ALS-associated SOD1 mutant protein induces changes in HSP70 and HSC70 client association and aggregation toward polypeptides with predicted disorder, indicating that there are global effects from a single misfolded protein that extend to many clients within chaperone networks. Together these findings show that the ubiquitin-mediated UBAIT fusion system can efficiently isolate the complex interactome of HSP chaperone family proteins under normal and stress conditions.

Comprehensive interactome profiling of the human Hsp70 network highlights functional differentiation of J domains

“…Hsp70s interact with thousands of client proteins, but each is present in minute quantities in relation to Hsp70. Other methods, such as the recently described ubiquitin-activated interaction traps (UBAITs) (Ryu et al, 2020), may be more appropriate for detecting such interactions.…”

Suboptimal Mitochondrial Activity Facilitates Nuclear Heat Shock Responses for Proteostasis and Genome Stability

“…S6B). We reason that nucleolar RAN may be one of the HSP70 clients under heat shock conditions (Ryu et al, 2020), possibly explaining MRPL2-depletion effects on NCT.…”

Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice

“…GLASS-ChIP was performed as described above, using no antibody samples as controls, in two biological replicates, except that the cells were crosslinked with formaldehyde for only 1 min. Samples eluted from the ProteinA/G beads were processed through a modified Filter-Assisted Sample Preparation protocol as previously described 58 . Protein identification by LC-MS/MS was provided by the University of Texas at Austin Proteomics Facility on an Orbitrap Fusion following previously published procedures 59 .…”

“…Samples eluted from the ProteinA/G beads were processed through a modified Filter-Assisted Sample Preparation protocol as previously described 58 . Protein identification by LC-MS/MS was provided by the University of Texas at Austin Proteomics Facility on an Orbitrap Fusion following previously published procedures 59 .…”