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. 2022 Oct 21;20(10):e3001839.
doi: 10.1371/journal.pbio.3001839. eCollection 2022 Oct.

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

Nitika  1 , Bo Zheng  1 , Linhao Ruan  2   3 , Jake T Kline  4 , Siddhi Omkar  1 , Jacek Sikora  5 , Mara Texeira Torres  6 , Yuhao Wang  2   3 , Jade E Takakuwa  1 , Romain Huguet  7 , Cinzia Klemm  6 , Verónica A Segarra  8 , Matthew J Winters  9 , Peter M Pryciak  9 , Peter H Thorpe  6 , Kazuo Tatebayashi  10   11 , Rong Li  2   3   12   13 , Luca Fornelli  4 , Andrew W Truman  1
Affiliations

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

Nitika et al. PLoS Biol. .

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
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.
Fig 2
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.
Fig 3
Fig 3. Direct interactors of yeast Hsp70 identified by XL-MS.
(A) Schematic representation of 177 inter protein cross-links and identified PTMs of direct interactors on domains of Hsp70. (B) Functional classification of direct Hsp70-protein peptides. (C) Western blot analysis of HA-tag immunoprecipitated Cct8, Pcl7, Ura8, and Sse1 from yeast cells. CTD, C-terminal domain; NBD, nucleotide-binding domain; PTM, posttranslational modifications; SBD, substrate-binding domain; XL-MS, cross-linking mass spectrometry.
Fig 4
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.
Fig 5
Fig 5. Activity of Pim1/Lonp-1 is regulated by interaction with Hsp70 and a novel phosphorylation site, S974.
(A) Schematic representation of Ssa1-Pim1 inter protein cross-links detected on NBD of Ssa1 and proteolytic domain of Pim1. (B) Ssa1-Pim1 cross-links mapped on the crystal structures of Pim1 and Hsp70. (C) Pim1 interacts with the chaperone complex in yeast cells. Yeast expressing Pim1-GFP were grown to mid-log phase and Pim1-GFP complexes were isolated using GFP-TRAP beads. The purified Pim1-GFP complex was analyzed by SDS-PAGE/western blot analysis using indicated antisera. (D) Lonp-1 interacts with chaperone complexes in mammalian cells. HEK293 cells were transfected with a FLAG-Lonp-1 construct and after 24 h, total protein was extracted and FLAG-Lonp-1 complexes were isolated using FLAG Dynabeads. FLAG-Lonp-1 complexes were analyzed by SDS-PAGE/western blot using indicated antisera. (E) Western blot analysis of Lonp-1 upon addition of Hsp70 inhibitor JG-98 and proteasomal inhibitor Bortezomib. HEK293 cells were grown to mid-confluence and were treated with the indicated reagents/times. Lysates were analyzed by SDS-PAGE/western blot using indicated antisera. (F) Growth assay of Pim1 phospho-site mutants in yeast. Indicated cells were grown under indicated conditions in 96-well format in a Synergy H1 plate reader. OD600 readings were taken at regular intervals for 35 min. Data shown are the average and standard deviation of at least 5 biological replicates. (G) Fluorescence images of cells expressing FlucSM–RFP and Tom70-GFP. Scale bars are 10 μm. (H) Western blot analysis of Pim1 wild type and phospho-mutants upon addition of Bortezomib. *Indicates Pim1 self-cleavage product. (I) IP analysis of Pim1 wild type and phospho-mutants with chaperone complex. Indicated cells were grown and processed as in (A). (J) Western blot analysis of Pim1 (wild type and phospho-mutants) expressed in the presence or absence of Ssa1 in E. coli. BL21 cells were co-transformed with indicated plasmids and were grown to early mid-log phase whereupon protein expression was induced with IPTG. After 4 h, total cell protein was isolated via sonication and lysates were analyzed by western blotting with indicated antisera. *Denotes Pim1 self-cleavage product. (K) Schematic of Ssa1 regulation of Pim1. Phosphorylated Pim1 interacts with Ssa1 preventing inappropriate activation of Pim1 in the cytoplasm. Pim1 dephosphorylation and Ssa1 dissociation permit Pim1 self-cleavage, critical for Pim1 activity in the mitochondria. Created with BioRender.com. The data underlying the graphs shown in the figure can be found in S1 Data. CTD, C-terminal domain; NBD, nucleotide-binding domain; SBD, substrate-binding domain.
Fig 6
Fig 6. Mtw1 is a client of Hsp70 and is regulated by phosphorylation.
(A) Schematic representation of Ssa1-Mtw1 inter protein cross-links detected on NBD of Ssa1 and head domain of Mtw1. (B) Ssa1-Mtw1 cross-links mapped on the crystal structure of Ssa1 and Mtw1. (C) Mtw1 interacts with the chaperone complex. Yeast expressing Mtw1-FLAG were grown to mid-log phase and Mtw1 complexes were isolated using FLAG dynabeads. The purified Mtw1 complex was analyzed by SDS-PAGE/western blot analysis using indicated antisera. (D) Mtw1 is destabilized in temperature-sensitive Ssa1-45 mutant strain. Indicated yeast were transformed with a plasmid expressing Mtw1-GFP. Yeast were grown to mid-log and were then were either left untreated or were exposed to heat shock at 39°C for 90 min. Levels of Mtw1 were assessed via western blot using antisera to indicated proteins. (E) Mis12 interacts with chaperone complexes in mammalian cells. HEK293 cells were transfected with a Mis12-GFP construct and after 24 h, total protein was extracted, and Mis12 complexes were isolated using GFP-TRAP beads. Mis12 complexes were analyzed by SDS-PAGE/western blot using indicated antisera. (F) Western blot analysis of Mis12 upon addition of Hsp70 inhibitor JG-98 and proteasomal inhibitor bortezomib. (G) Growth assay analyzing the phenotype of the Mtw1 and its Y86 mutant. (H) Mtw1 was tagged with YFP in wild-type and Mtw1-Y86F mutant strains to compare Mtw1 localization at the kinetochore using fluorescence microscopy. (I) Fluorescence intensities were quantified in wild-type and mutant Mtw1 using the semi-automated FociQuant ImageJ script [92]. Intensities were compared using the Student t test (p-value = 1.8 × 10−12). (J) Analysis of the impact of Mtw1 phosphorylation on interaction with chaperones. (K) Model of Mtw1 activity regulation via its phosphorylation. Dephosphorylation of Mtw1 promotes dephosphorylation and correct association with kinetochore components such as Mif2 and Nnf1. Created with BioRender.com. The data underlying the graphs shown in the figure can be found in S1 Data. CTD, C-terminal domain; NBD, nucleotide-binding domain; SBD, substrate-binding domain.
Fig 7
Fig 7. Ste11 dimethylation impacts the osmotic stress response.
(A) Depiction of Ste11 pathway. (B) Schematic representation of Ssa1-Ste11 inter protein cross-links detected on NBD of Ssa1 and regulatory domain of Ste11. (C) Halo assay analyzing the phenotype of the Ste11 wild-type and methylation mutants in response to alpha factor. (D) FUS1-lacZ activity of Ste11 mutants in response to pheromone. Indicated yeast were grown to mid-log phase and then were processed as in [52]. (E) Western blot analysis of the effect of Ste11 wild type and mutants in response to pheromone signaling. Indicated cells were grown to mid-log phase, treated with the indicated quantity of alpha factor. Phosphorylation of Fus3 was measured by analysis of lysates via western blotting with indicated antisera. (F) Growth assay of Ste11 methylation mutants in response to hyperosmotic stress. Cells were grown to mid-log phase, were 10-fold serially diluted and then plated onto appropriate media using a 48-pin replica-plating tool. Images of plates were taken after 3 days at 30°C. (G) 8xCRE-lacZ activity of Ste11 mutants in response to osmotic stress. Yeast transformed with the 8xCRE lacZ reporter were grown to mid-log phase and then treated with the indicated stressors. Cell lysate was extracted and beta-galactosidase activity was measured as in [50]. Data shown are the mean and standard deviation of at least 5 biological replicates. (H) Halo assay showing that none of the Ste11 variants permit pheromone response in the absence of Ste20. (I) Impact of Ste11 methylation on chaperone interactions. Yeast transformed with indicated HA-Ste11 constructs were grown to mid-log phase and Ste11 complexes were isolated using HA-magnetic beads. The purified Ste11 complexes were analyzed by SDS-PAGE/western blot analysis using indicated antisera. (J) Model of methylation-dependent regulation of Ste11 activity regulation. Di-methylation of Ste11 selectively alters osmotic signaling but not mating pathway activity. R405 di-methylation may alter Ste11 association with key osmotic signaling proteins such as Ste50. Created with BioRender.com. The data underlying the graphs shown in the figure can be found in S1 Data. CTD, C-terminal domain; NBD, nucleotide-binding domain; SBD, substrate-binding domain.
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