Summary
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 inability of current technologies to distinguish direct vs bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the 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 multi-point interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors which can be used to probe chaperone function in cells, we have also identified a suite of 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.
In vivo confirmation of Hsp70 dimerization
Comprehensive direct interactome of Hsp70
Multi-domain interactions between Hsp70 and client proteins
Identification of novel biologically-important client protein PTMs
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- AP-MS
- Affinity purification mass spectrometry
- BiFC
- Bimolecular fluorescence complementation
- CMV
- human cytomegalovirus promoter
- CTD
- C-terminal Domain
- DSSO
- disuccinimidyl sulfoxide
- GO
- Gene ontology
- HSE
- Heat Shock Response Element
- HSP
- Heat Shock Protein
- MS
- mass spectrometry
- NBD
- Nucleotide-binding domain
- PTM
- Post-translational modifications
- SBD
- Substrate-binding domain
- VN
- Venus amino-terminal end
- VC
- Venus carboxy-terminal end
- XL-MS
- Cross-linking mass spectrometry
- Y2H
- Yeast two-hybrid