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Comprehensive characterization of the Hsp70 interactome reveals novel client proteins and interactions mediated by posttranslational modifications [1]

['Department Of Biological Sciences', 'The University Of North Carolina At Charlotte', 'Charlotte', 'North Carolina', 'United States America', 'Bo Zheng', 'Linhao Ruan', 'Center For Cell Dynamics', 'Department Of Cell Biology', 'Johns Hopkins University School Of Medicine']

Date: 2022-11 

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.

Funding: This work was supported by the NIH (R15GM139059 and R01GM139885 to AWT, R01GM057769 to PMP), the Queen Mary University of London and the Francis Crick Institute (Cancer Research UK—FC001183; UK Medical Research Council—FC001183 and the Wellcome Trust—FC001183 to PHT), a grant from Re-Stem Biotech to R.L., the JSPS Grants-in-Aid for Scientific Research (KAKENHI) (21H02422 to KT), the Institute for Fermentation, Osaka (G-2021-2-082 to KT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Importantly, Hsp70 stabilizes and activates of a wide range of signaling molecules including those involved in processes such as DNA damage response, cell cycle control, autophagy, and nutrient sensing [ 10 , 17 – 19 ]. The Hsp70 client proteins involved in these cellular processes tend to be either highly posttranslationally modified (PTMs) or regulate PTMs on other proteins. In turn, these PTMs tightly regulate a multitude of protein properties including subcellular localization, enzymatic activity, and protein interactions [ 20 ]. Advances in mass spectrometry-based methods have allowed the identification of more than 200 different types of PTMs on proteins including phosphorylation, acetylation, and ubiquitination [ 21 – 23 ]. Given the numerous PTMs identified on proteins, researchers are now facing difficult choices when selecting specific PTMs for further study. Computational methods for identifying important PTMs on proteins have been partially successful but rely on preexisting MS data [ 21 , 24 ]. In this report, we have utilized XL-MS to comprehensively understand the Hsp70 interactome. In doing so, we have uncovered not only a new set of interactors that directly bind to Hsp70, but also show that these proteins bind at multiple sites on Hsp70, including the N-terminal NBD. Notably, many of the Hsp70 interactions are in close proximity to novel biologically important PTMs. 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 proteins.

The essential nature of Hsp70 function in the cell, as well as its involvement in a variety of human pathologies such as cancer, has driven researchers to set out to characterize Hsp70 interactors. While great strides have been made towards this goal, these efforts have been hampered by limitations in the technologies used. For example, these past efforts have utilized affinity purification followed by mass spectrometry (AP-MS), yeast two-hybrid (Y2H), and proximity proteomics methodologies, all of which lack the ability to discriminate between direct and bridged protein interactions [ 7 – 12 ]. Chemical cross-linking with mass spectrometry (XL-MS) is a powerful interactomic technique that circumvents this issue, providing information on direct interactions in protein complexes by using chemical cross-linkers [ 13 – 16 ]. Indeed, XL-MS studies are often complementary to traditional structural biology methods such as X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy [ 16 ].

The maintenance of a correctly folded proteome (proteostasis) is critical for cell survival. Cells maintain proteostasis under both basal and stress conditions through the expression of molecular chaperones such as Hsp70 and its associated co-chaperone regulators [ 1 , 2 ]. Hsp70 function is dependent on 3 conserved domains: an N-terminal nucleotide-binding domain (NBD), a substrate (“client”)-binding domain (SBD), and a C-terminal “lid” domain (CTD) [ 1 , 2 ]. The binding and hydrolysis of ATP to ADP in the NBD promotes large-scale structural Hsp70 rearrangements that allow the closing of the CTD over client proteins that bind in the SBD, promoting protein folding [ 1 , 3 ]. The characterized roles of Hsp70 include folding of new and denatured proteins, transport of mitochondrial proteins and disaggregation of protein complexes [ 4 – 6 ].

Results

Analysis of cross-linked yeast Hsp70 complexes Previous studies have identified proteins in complex with yeast Hsp70 (Ssa1) using quantitative AP-MS [9]. To comprehensively identify novel direct Ssa1 interactors along with their associated points of interaction, we took a novel cross-linking proteomics approach. HIS-tagged Ssa1 was expressed in ssa1-4Δ, a yeast strain in which all 4 SSA (Hsp70) genes have been deleted [25]. HIS-Ssa1 complexes were cross-linked with disuccinimidyl sulfoxide (DSSO), an MS-cleavable cross-linker [26]. These complexes were digested into peptides via trypsin and were characterized via mass spectrometry (see Fig 1A). The DSSO cross-links present on the cross-linked peptides were cleaved by collision-induced dissociation during the MS2 stage of mass spectrometry, allowing analysis and identification of each of the single peptide chains at the MS3 level as in [26]. Overall, we identified 1,510 interactors associated with HIS-Ssa1 in the cross-linked complexes and 1,152 in control HIS-Ssa1 complexes without DSSO-mediated cross-linking (Fig 1B). We anticipated that proteins present in the DSSO-treated complexes may consist of direct Ssa1 interactors including client proteins and co-chaperones. To distinguish direct interactors of Ssa1 from indirect interactors, we filtered our data for cross-linked peptides where at least one of the identified peptides was Ssa1. We identified a total of 363 Ssa1-containing cross-linked peptides, out of which 177 were Ssa1-interactor cross-links and 106 were Ssa1-Ssa1 cross-links (Fig 1C). Validating our methodology, no cross-linked peptides were observed in the control uncross-linked sample. To determine whether the cross-linking process had enriched for any particular class of protein, we performed Gene Ontology (GO) analysis of unique candidate interactors of cross-linked and control samples. This GO analysis revealed enrichment of multiple cellular functions (Fig 1D). In the cross-linked samples, proteins involved in protein folding, trafficking, and cell signaling were all enriched, consistent with the established roles of Hsp70. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Cross-linking mass spectrometry of Ssa1 complexes. (A) Experimental workflow of cross-linking mass spectrometry of Ssa1 complexes purified from yeast cells. HIS-Ssa1 complexes were cross-linked with DSSO, purified from yeast, digested into peptides via trypsin, and analyzed by mass spectrometry. Created with BioRender.com. (B) Venn diagram representing Ssa1 complexes found in conventional IP and DSSO-treated IP. (C) Pie chart showing types of cross links identified from XL-MS analysis. (D) GO analysis of DSSO-treated Ssa1 immunoprecipitated complexes and cross-linked Ssa1 complexes using TheCellMap.org. DSSO, disuccinimidyl sulfoxide; GO, Gene Ontology; XL-MS, cross-linking mass spectrometry. https://doi.org/10.1371/journal.pbio.3001839.g001

Ssa1 oligomerization is required for a subset of chaperone functions Nearly a third of the cross-linked peptides detected in our experiment were between 2 Ssa1 peptides (Fig 1C). To distinguish whether the cross-linked Ssa1-Ssa1 peptides came from dimerized Ssa1 molecules as opposed to single intramolecular cross-links, we initially mapped the identified cross-links onto homology-based Ssa1 models. Given the large conformational change Hsp70 undergoes during its folding cycle, we mapped obtained cross-links onto both ADP-bound (“open”) and ATP-bound (“closed”) structures of Hsp70 (Figs 2A and S1A). The mapping of cross-links onto these models revealed that a substantial number of Ssa1-Ssa1 peptides had cross-linking lengths well within the spacer arm limit for DSSO, while many others exceeded the lengths possible by cross-linking within a single molecule, implying potential dimerization (Figs 2B and S1A). Dimers of Hsp70 from different organisms have previously been observed [27] and we decided to evaluate whether the same was true for yeast Ssa1. To visualize Ssa1-Ssa1 interaction in live yeast, we utilized bimolecular fluorescence complementation (BiFC). Yeast expressing Ssa1 tagged with Venus amino-terminal end (VN) and Venus carboxy-terminal end (VC) were examined using high-resolution fluorescence microscopy. Imaging of these cells revealed that Ssa1 dimers were clearly visible and that they localized primarily to the nucleus, whereas no BiFC signal was observed in cells expressing only VN or VC-Ssa1 (Figs 2C and S1B). Previous structural studies of both the DnaK dimer in bacteria and Hsc70 dimer in mammalian cells identified 2 key residues important in dimer formation, N537 and D540 [28,29]. Mutation of these sites on DnaK (N537A and D540A) results in bacteria that are heat sensitive [28]. We created equivalent mutations in Ssa1 (N537A/E540A) and assessed the ability of WT and the mutant to interact via co-immunoprecipitation (Fig 2D). Similar to previous studies using bacterial DnaK or mammalian Hsc70, the ability of N537A/E540A mutant to interact with WT Ssa1 was substantially compromised (Fig 2D). To demonstrate in vivo functionality of the Ssa1 dimer, we examined the ability of N537A/E540A to support viability and the cellular response to heat. While yeast cells expressing the N537A/E540A dimer-deficient mutant were viable and grew at approximately WT rates, they were impaired for growth at high temperature (Fig 2E). The temperature-sensitive phenotype of N537A/E540A was suppressed by osmotic stabilization, suggesting impairment of the heat shock response and cell integrity signaling (Fig 2E). To further explain this temperature-sensitive phenotype, we assessed the activity of the heat shock response in WT and N537A/E540A cells using a real-time destabilized luciferase reporter [30]. WT cells produced a robust heat shock response element (HSE)-luciferase signal after heat exposure, whereas N537A/E540A cells did not (Fig 2F). To complement our HSE-reporter result, we determined the impact of N537A/E540A on the induction of 2 well-characterized heat-inducible proteins, Hsp26 and Hsp42. Hsp26 and Hsp42 levels did not respond to exposure to 39°C in N537A/E540A cells, confirming that loss of Ssa1 self-association impacts the heat shock response (Fig 2G). Insofar as Ssa1 is a major hub for protein folding in yeast, we set out to examine the possibility that some of the observed Ssa1-Ssa1 interactions might be the result of active Ssa1 folding a newly synthesized Ssa1 polypeptide chain. We studied the interactions of FLAG-Ssa1 (WT and substrate-binding deficient mutant V435F) with a known client, Rnr2 [9], GAPDH, a known interactor but not a client of Ssa1 in yeast [11], the Ydj1 co-chaperone, and HA-Ssa1. Although WT Ssa1 co-purified with GAPDH, Rnr2, Ydj1, and Ssa1, the V435F mutant maintained only interaction with GAPDH Ydj1 and Ssa1, demonstrating that Ssa1 is not a client of other Ssa1 molecules (Fig 2H). Taken together, these findings confirm that Ssa1 dimerizes in yeast and that this self-interaction is important for a subset of Ssa1 functions. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Ssa1 oligomerization is required for a fully functional heat shock response. (A) Identified Ssa1-Ssa1 cross-links mapped on the domains of Ssa1 in open and closed conformation. (B) Ssa1-Ssa1 cross-links that exceeded the DSSO spacer arm length when mapped to the monomeric structure of Ssa1 and thus potentially represent interactions between different Ssa1 molecules (dimers or oligomers). (C) Fluorescence images of diploid cells expressing both of the N-terminally VN- and VC-tagged versions of Ssa1. DAPI was used as a nuclear marker and the scale bars are 10 μm. (D) Analysis of Ssa1-Ssa1 interactions in yeast via co-immunoprecipitation. Ssa1-4Δ cells transformed with plasmids expressing GFP-Ssa1 and FLAG-Ssa1 were grown to mid-log phase. After extraction of total protein, FLAG-tagged Ssa1 complexes were purified via FLAG-magnetic beads and analyzed via SDS-PAGE/western blotting with indicated antisera. (E) Serial dilution of yeast expressing mutations that impact Ssa1-Ssa1 interactions. Yeast strains were grown to mid-log phase and then 10-fold serially diluted onto YPD media at the indicated conditions. Plates were photographed after 3 days. (F) Real-time luciferase reporter assay of Hsf1 activity over a 200-min heat shock at 39°C. Indicated yeast strains were transformed with a real-time luciferase reporter (HSE-lucCP+) and were processed as in [30]. The data shown are the average and standard deviation of at least 5 biological replicates. (G) Inducibility of Hsp26 and Hsp42 in WT and N537A/E540A yeast. Cells were grown to mid-log at 25°C and were then shifted to 39°C for 90 min. Protein lysate from these samples were analyzed by SDS-PAGE followed by western blotting using antisera for Hsp26, Hsp42, and Pgk1. (H) Western blot analysis of Flag-Ssa1 complexes (WT and V435F mutant) purified from cells expressing HA-tagged Ssa1. The data underlying the graphs shown in the figure can be found in S1 Data. CTD, C-terminal domain; DSSO, disuccinimidyl sulfoxide; NBD, nucleotide-binding domain; SBD, substrate-binding domain; VC, Venus carboxy-terminal end; VN, Venus amino-terminal end. https://doi.org/10.1371/journal.pbio.3001839.g002

The HIR complex is a novel client of Hsp70 The histone regulator (HIR) protein complex regulates histone gene transcription, nucleosome formation, and heterochromatic gene silencing [31,32]. Our XL-MS analysis revealed a novel direct interaction between Ssa1 and HIR complex components Hir1 and Hir2. We observed cross-linking between the SBD of Ssa1 and residues K435 of Hir1 and K452 of Hir2, adjacent to their respective nuclear localization signals (Fig 4A and 4B). To validate our XL-MS finding, we carried out Co-IP followed by immunoblotting of Hir1 and Hir2 with key chaperone components Ssa1, Sse1, Hsp82, and Ydj1. These experiments confirmed a strong association between HIR and the chaperones tested (Fig 4C). Furthermore, we confirmed that Hir1 and Hir2 are bona fide Ssa1 client proteins by demonstrating that loss of Ssa1 function resulted in Hir1 and Hir2 destabilization (Fig 4D). PPT PowerPoint slide

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TIFF original image Download: Fig 4. HIR complex is a novel client of Hsp70 in yeast and humans. (A) Schematic representation of Ssa1-Hir1/Hir2 inter protein cross-links detected on SBD of Ssa1 and NLS of Hir1 and NTD of Hir2. (B) Ssa1-Hir1/2 cross-links mapped on the crystal structure of Ssa1, Hir1, and Hir2. (C) The Hir complex interacts with yeast chaperones. (D) Hir1 and Hir2 are dependent on Ssa1 for their stability. Indicated yeast cells were transformed with plasmids expressing HA-Hir1 or HA-Hir2 driven via the GAL1 promoter. Yeast were grown to mid-log in YPGalactose-URA media and then were either left untreated or were exposed to heat shock at 39°C for 90 min. Levels of Hir1 or Hir2 were assessed via western blot using antisera to indicated proteins. (E) HIRA interacts with chaperone complexes in mammalian cells. HEK293 cells were transfected with an HA-HIRA construct. After 24 h, total protein was extracted and HIRA complexes were purified via HA-magnetic beads. The purified HIRA complexes were analyzed by SDS-PAGE followed by western blotting using the indicated antisera. (F) Western blot analysis of HIRA upon addition of Hsp70 inhibitor JG-98 and proteasomal inhibitor bortezomib. CTD, C-terminal domain; HIR, histone regulator; NBD, nucleotide-binding domain; SBD, substrate-binding domain. https://doi.org/10.1371/journal.pbio.3001839.g004 To demonstrate evolutionary conservation of the identified chaperone-HIR interaction, we examined the interaction between human Hsc70 and HIRA, the major HIR complex protein in human cells. Consistent with our results in yeast, HA-HIRA co-immunoprecipitated with Hsc70, Hsp110, Hsp90, DNAJA1 (Fig 4E). To examine the dependence of HIRA on Hsc70 chaperone activity, we treated HEK293 cells with the Hsp70 inhibitor JG-98 and monitored HIRA abundance over time. HIRA levels rapidly decreased after JG-98 addition, with HIRA becoming undetectable after 2 h (Fig 4F). Given that in our system HA-HIRA was expressed under the constitutive human cytomegalovirus (CMV) promoter, we hypothesized that the effect we observed on HIRA abundance could be explained by protein degradation. Supporting this hypothesis, addition of the proteasomal inhibitor bortezomib prevented JG-98-dependent HIRA loss (Fig 4F). Taken together, our results suggest that HIR complex proteins are client proteins of the Hsp70 chaperone system in yeast and mammalian cells.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001839

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