Abstract
The 70 kDa heat shock protein (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 71 kDa heat shock cognate (HSC70) clients using a ubiquitin-mediated proximity tagging strategy and show that, despite their high degree of similarity, these enzymes have largely nonoverlapping 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 amyotrophic lateral sclerosis (ALS)-associated superoxide dismutase 1 (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-activated interaction trap (UBAIT) fusion system can efficiently isolate the complex interactome of HSP chaperone family proteins under normal and stress conditions.
Highlights
Every cell has a finely tuned balance of protein expression, folding, complex formation, localization, and degradation that is specific to its growth state and environmental cues
We find that HSP70 and HSC70 are largely nonoverlapping for client association under normal growth conditions, both chaperones show a bias toward binding to newly synthesized polypeptides
One of the primary advantages of this system is that interacting proteins accumulate as covalent ubiquitination conjugates to the bait protein, so that even transient binding partners can be readily identified by mass spectrometry
Summary
Every cell has a finely tuned balance of protein expression, folding, complex formation, localization, and degradation that is specific to its growth state and environmental cues. Many pathological states involve changes in this balance, resulting in loss of regulatory control or loss of resilience during stress [1,2]. While gene expression is quantitatively measured at the level of nucleic acids and polypeptides, determining the status of cells with respect to protein homeostasis is still a challenge, as we lack the tools to examine folding of individual proteins and the functionality of multicomponent complexes quantitatively on a global scale. The front line of defense against challenges to protein homeostasis is composed of cellular chaperones—proteins that recognize unfolded, aggregated, or unstable polypeptides [3].
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