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Cellular responses to proteostasis perturbations reveal non-optimal feedback in chaperone networks

  • Asmita Ghosh
  • Abhilash Gangadharan
  • Monika Verma
  • Sarada Das
  • Latika Matai
  • Devi Prasanna Dash
  • Debasis Dash
  • Koyeli Mapa
  • Kausik ChakrabortyEmail author
Original Article
  • 120 Downloads

Abstract

The proteostasis network (PN) comprises a plethora of proteins that are dedicated to aid in protein folding and maintenance; some with overlapping functions. Despite this, there are multiple pathophysiological states associated with depletion of chaperones. This is counter-intuitive, assuming cells have the ability to re-program transcriptional outputs in accordance with its proteostasic limitations. Here, we have used S. cerevisiae to understand how cells respond to different types of proteostasis impairments. We monitored the proteostasis status and transcriptome of single deletions of fourteen different Protein Quality Control (PQC) genes. In most cases, cellular response did not activate proteostasis components or pathways that could either complement the function of the missing PQC gene or restore proteostasis. Over-expression of alternate machineries could restore part of the proteostasis defect in two representative PQC gene deletion strains. We posit that S. cerevisiae inherently lacks the ability to sense and respond optimally to defects in proteostasis caused due to deletion of specific PQC components.

Keywords

Chaperones Transcriptomics Epistasis Proteostasis S. cerevisiae 

Notes

Acknowledgements

We are grateful to Dr. Mohammed Faruq for aiding us with the Illumina sequencing platform. We thank Dr. Deepak Sharma (affiliated to CSIR-IMTECH) for assistance with reagents. This work was primarily funded by OLP1104 grant by CSIR to KC and partially by the grant YSS/2015/000532 from SERB to KM along with SNU core funding. We thank the HPC facility of CSIR-IGIB, for aiding us with computing resources. AG1 (Asmita), AG2 (Abhilash) and LM thank UGC for their fellowship. MV is grateful to CSIR, SD to SNU-core funding, and DPD to DBT for their fellowships.

Author contributions

AG1 and KC designed the work. KC, KM and DD supervised the work and analysis. Sequencing was done by AG1. LM made the TS mutants of Nat-R. DPD made Mutants of yeGFP. AG1, SD, MV did the yeast experiments. The transcriptomics experiments were done by AG1. Analysis was primarily done by AG2 along with AG1 and KC. AG1 and KC wrote the manuscript with input from all authors. All authors read and approved the final version of the manuscript.

Compliance with ethical standards

Conflict of interest

Authors declare no competing interests.

Supplementary material

18_2019_3013_MOESM1_ESM.pdf (3.6 mb)
Supplementary Figure S1 (A) Representative blots for cycloheximide chase of TS 22 and WT Nat are shown. Ponceau was used for loading normalisation. (B) Growth curve of Wt Nat and TS22 at 30 °C done on Bioscreen. Average of 3 biological replicates is plotted. (C) Steady state amounts of Wt Nat and TS22 upon 20 µM celastrol treatment at 30 °C were determined by western blot analysis (n = 3). Representative blot shown. (D) Representative blots for steady state amounts of Wt Nat at 30 °C in different chaperone deletion strains were determined by western blot analysis. Ponceau was used for loading normalisation. (E) Quantification from 3 biological replicates are plotted for WT Nat in different PQC gene deletion strains. (F) Amounts of TS22 normalized to the amount of WT Nat in the respective chaperone deletion strains are quantitated (n = 3). (G) Growth (at 30 °C) corresponding to the activity of TS22 in PQC deletion strains when grown in different concentrations of cloNat in shown. The error bars are the standard deviation from three biological replicates. The left panel is for rqc2Δ, the middle panel is for sse1Δ and the right panel is for hsp82Δ. Wt Nat in each corresponding strain acts as the control (PDF 3686 kb)
18_2019_3013_MOESM2_ESM.pdf (151 kb)
Supplementary Figure S2 A) HSE-GFP reporter levels in these PQC deletions at 25 °C and 37 °C as obtained fromBrandman et al. [24]. B) Expression levels of HSF1-inducible ssa4 and PKA-inducible hsp12 mRNA are shown for the different PQC deletions. C) The panel shows the expression of canonical chronic HSR (stressed for 6 h at 37 °C) genes in different PQC gene deletions. The 37 °C HSR column is the positive control which shows these genes are highly upregulated upon chronic HSR. The fold change in the expression of these genes in PQC deletion strains w.r.t BY4741 (parental strain) was compared to the global alteration in transcripts using Mann–Whitney test. The p values obtained were corrected for multiple comparisons using FDR and the genes in any of these strains that did not cross the cut-off significance for p value 0.05 are without any colour (blank boxes). The colour scheme is graded from red (down-regulated) to blue (upregulated) showing median fold change values. The size of the circle represents the median fold change shift in either direction (PDF 150 kb)
18_2019_3013_MOESM3_ESM.pdf (4.4 mb)
Supplementary Figure S3 A) For each pair of the PQC deletions transcriptome alteration was quantified w.r.t WT strain. Pairwise comparison was then done between the different PQC deletions using transcriptome alterations. The upper triangular matrix shows the Pearson correlation between the PQC deletions. Lower triangular part of the graph is scatter plots of the transcriptomes of the deletion pairs. The trend lines are LOESS fits and are guides for visualization. B) Transcriptional alteration of each gene (fold change in a PQC gene deletion with respect to WT strain from this study) is plotted against their Boone epistasis score (ε) as reported (27) (PDF 4477 kb)
18_2019_3013_MOESM4_ESM.pdf (6.2 mb)
Supplementary Figure S4 A) BY4741 and sse1Δ bearing either empty vector or galactose-inducible over expression of Sse1 or Sse2 were spotted on YPD, YP + Raffinose or YP + Galactose and grown at 30 °C. B) BY4741 and jjj1Δ bearing either empty vector or galactose-inducible over expression of Jjj1 or Asc1 or Vam7 or Vid22 were spotted on YPD, YP + Raffinose or YP + Galactose and grown at 30 °C. C) Representative blot showing steady state amounts of TS22 at 30 °C in By4741 and jjj1Δ with or without 2.5 mM paraquat treatment. D) Upper graph corresponds to activity, measured by growth in different concentrations of Clonat, of Wt Nat in jjj1Δ in the presence and absence of paraquat. The lower graph corresponds to activity of Wt Nat in BY4741 in the presence and absence of paraquat. Error bars are from standard deviation of three biological replicates (PDF 6298 kb)
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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Asmita Ghosh
    • 1
    • 2
  • Abhilash Gangadharan
    • 1
    • 2
  • Monika Verma
    • 1
    • 2
  • Sarada Das
    • 3
  • Latika Matai
    • 1
    • 2
  • Devi Prasanna Dash
    • 1
    • 2
  • Debasis Dash
    • 1
    • 2
  • Koyeli Mapa
    • 3
  • Kausik Chakraborty
    • 1
    • 2
    Email author
  1. 1.CSIR-Institute of Genomics and Integrative BiologyDelhiIndia
  2. 2.Academy of Scientific and Innovative Research, Coordination OfficeCSIR-Human Resource Development Centre CampusGhaziabadIndia
  3. 3.Department of Life Sciences, School of Natural SciencesShiv Nadar UniversityGreater NoidaIndia

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