Comprehensive characterization of the Hsp70 interactome reveals novel client proteins and interactions mediated by posttranslational modifications

Abstract

Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and the inability of current technologies to distinguish direct versus bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the Saccharomyces cerevisiae (budding yeast) Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multipoint interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors that can be used to probe chaperone function in cells, we have also identified a suite of posttranslational modification (PTM)-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically important PTMs on client proteins.

Editorial: Guardians of protein homeostasis (proteostasis) in health, disease and aging

“…Although the molecular details of these interactions are yet to be fully understood, the review highlights the emerging features of these proteins and their interaction with Hsp70s. The study underscores the versatile functions of Hsp70 chaperones and expands on recent discoveries demonstrating that Hsp70 interacts with numerous proteins throughout its domains and that those interactions are regulated by post-translational modifications ( Nitika et al, 2022 ).…”

Metabolic regulation of misfolded protein import into mitochondria

“…Three possible factors could therefore contribute to the reduced accumulation of MPs in mitochondria of Snf1-active cells: 1) enhanced intra- mitochondrial degradation, 2) reduced cytosolic misfolded protein (due to enhanced folding and/or other degradation pathways), and 3) blocked mitochondrial import (Figure 2A). To evaluate the first possibility, an antimorphic mutant pim1 S974D was used to block the degradation of imported FlucSM in the mitochondrial matrix ( 23 ). Indeed, in HG medium wild-type cells overexpressing pim1 S974D showed a significantly increased accumulation of FlucSM in mitochondria compared to cells overexpressing PIM1 (Figure 2B and 2C).…”

Second international symposium on the chaperone code, 2023

“…Taken together, his results suggest that Hsp70 acts as a major integrator of diverse cellular signals. 26 , 27 Lila Gierasch (University of Massachusetts-Amherst, USA) focused on the finding that some substrate peptide sequences bound to Hsp70 in two orientations, C- to N- or N- to C-, suggesting that the preferred binding mode is a result of the optimal positioning of substrate side chains in the binding cleft regardless of the backbone orientation. 28 She presented new findings on a peptide designed with a “palindromic sequence” that presents the same side chain sequences around the residue that occupies the “central pocket” regardless of the binding orientation.…”