RT Journal Article
SR Electronic
T1 A novel multifunctional role for Hsp70 in binding post-translational modifications on client proteins
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.08.25.457671
DO 10.1101/2021.08.25.457671
A1 Nitika
A1 Bo Zheng
A1 Linhao Ruan
A1 Jake T. Kline
A1 Jacek Sikora
A1 Mara Texeira Torres
A1 Yuhao Wang
A1 Jade E. Takakuwa
A1 Romain Huguet
A1 Cinzia Klemm
A1 Verónica A. Segarra
A1 Matthew J. Winters
A1 Peter M. Pryciak
A1 Peter H. Thorpe
A1 Kazuo Tatebayashi
A1 Rong Li
A1 Luca Fornelli
A1 Andrew W. Truman
YR 2021
UL http://biorxiv.org/content/early/2021/08/25/2021.08.25.457671.1.abstract
AB 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 dimerizationComprehensive direct interactome of Hsp70Multi-domain interactions between Hsp70 and client proteinsIdentification of novel biologically-important client protein PTMsCompeting Interest StatementThe authors have declared no competing interest.AP-MSAffinity purification mass spectrometryBiFCBimolecular fluorescence complementationCMVhuman cytomegalovirus promoterCTDC-terminal DomainDSSOdisuccinimidyl sulfoxideGOGene ontologyHSEHeat Shock Response ElementHSPHeat Shock ProteinMSmass spectrometryNBDNucleotide-binding domainPTMPost-translational modificationsSBDSubstrate-binding domainVNVenus amino-terminal endVCVenus carboxy-terminal endXL-MSCross-linking mass spectrometryY2HYeast two-hybrid