Abstract 3917: A novel multifunctional role for Hsp70 in binding post-translational modifications on client proteins

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 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. In doing so, we have uncovered not only a new set of Hsp70 client proteins but show that these clients bind at multiple sites on Hsp70, including the N-terminal NBD. Notably, many of the Hsp70 interactions with client proteins are in close proximity to biologically important post translational modifications (PTMs). 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. All in all, our data suggest that our XL-MS approach to chaperone interactome characterization can also be used as a novel way to identify biologically important and previously unknown PTMs on client proteins. Citation Format: Nitika, Jake Kline, Luca Fornelli, Andrew Truman. A novel multifunctional role for Hsp70 in binding post-translational modifications on client proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3917.