Hsp70-associated chaperones have a critical role in buffering protein production costs
- Biological Research Centre of the Hungarian Academy of Sciences, Hungary
- Bay Zoltán Nonprofit Ltd, Hungary
Figures
Figure 1 with 1 supplement
Genetic perturbation analyses.
(A) Protein burden changes with copy number. The bar plot shows the relative fitness of yEVenus overexpressing and control genotypes as a function of plasmid copy number, a proxy of gene expression …
Figure 1—figure supplement 1
Additional analyses of the genetic interaction screen.
(A) Scatterplot of the genetic interaction scores and biomass-normalized fluorescence levels using knock-out strains with wild type cell size only. A prior systematic study (Jorgensen et al., 2002) …
Figure 2
Environmental screens under protein burden.
(A) Impact of translation inhibition on protein burden in wild type yeast. The bar plot shows the cost of yEVenus in wild type strain as a function of increasing cycloheximide concentration. …
Figure 3 with 1 supplement
Link between protein burden and proteotoxic stress.
(A) Impact of heat stress on protein burden. The bar plot shows the fitness cost of yEVenus in wild type strain as a function of increasing temperature. Protein burden significantly increased with …
Figure 3—figure supplement 3
Additional investigation of protein burden across different yEVenus plasmids and different chaperone-deficient mutants.
(A) Monitoring yEVenus fluorescence intensity in wild type cells across five different yEVenus plasmids. The figure shows fluorescence histograms of wild type cells with different plasmids. The …
Figure 4
Overview of the proteostasis network.
Malfunction of the protein quality control system impairs the proteome balance by driving cellular proteins into toxic metastable (partially folded or misfolded) conformations from their correctly …
Tables
Table 1
Functional enrichment analysis of genes showing synergistic interactions with yEVenus overexpression.
At an ε = - 0.05 cutoff value (and using a p<0.05 cutoff based on bootstrapping), 184 genes showed negative interactions with yEVenus. This gene set was tested for GO Slim category enrichment. A GO …
| Go.id | Term | N | Odds ratio | P value | FDR corrected P value |
|---|---|---|---|---|---|
| GO:0002181 | cytoplasmic translation | 11 | 2.27 | 1.52E-02 | 1.69E-01 |
| GO:0006325 | chromatin organization | 15 | 1.70 | 4.65E-02 | 2.93E-01 |
| GO:0006353 | DNA-templated transcription, termination | 3 | 4.17 | 4.60E-02 | 2.93E-01 |
| GO:0006354 | DNA-templated transcription, elongation | 16 | 8.43 | 2.73E-09 | 2.73E-07 |
| GO:0006360 | transcription from RNA polymerase I promoter | 6 | 5.86 | 1.24E-03 | 3.10E-02 |
| GO:0006366 | transcription from RNA polymerase II promoter | 29 | 2.29 | 2.01E-04 | 6.71E-03 |
| GO:0006397 | mRNA processing | 9 | 2.84 | 7.77E-03 | 1.11E-01 |
| GO:0006414 | translational elongation | 5 | 3.31 | 2.45E-02 | 2.34E-01 |
| GO:0006457 | protein folding | 10 | 3.06 | 3.26E-03 | 5.43E-02 |
| GO:0007005 | mitochondrion organization | 28 | 2.59 | 4.13E-05 | 2.07E-03 |
| GO:0009408 | response to heat | 7 | 3.17 | 1.05E-02 | 1.31E-01 |
| GO:0009451 | RNA modification | 7 | 2.53 | 2.91E-02 | 2.34E-01 |
| GO:0016570 | histone modification | 8 | 2.33 | 3.05E-02 | 2.34E-01 |
| GO:0032543 | mitochondrial translation | 12 | 2.96 | 1.78E-03 | 3.55E-02 |
| GO:0048308 | organelle inheritance | 5 | 3.23 | 2.68E-02 | 2.34E-01 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain (Saccharomyces cerevisiae) | Y7092 | PMID: 16118434 | SGA query strain, mat alpha | |
| Other | non-essential gene deletion collection (BY4741, MATa) | PMID:12140549 | YSC1053 | Open BioSystem (Dharmacon) |
| Other | synthetic genetic array (SGA) technique | PMID: 16118434 | ||
| Software, algorithm | ImageJ software | PMID: 22930834 | RRID:SCR_003070 | |
| Software, algorithm | Gene Onthology term enrichment with topGO (version 2.28) | PMID: 16606683 | ||
| Software, algorithm | org.Sc.sgd.db (version 3.3.0) packages in R | Core Team, 2017 | http://www.R-project.org | |
| Software, algorithm | Machine learning-based phenotypic analysis | PMID: 21807964 | ||
| Software, algorithm | Advanced Cell Classifier | PMID: 28647475 | http://www.cellclassifier.org/ | |
| Software, algorithm | Proteome Discoverer (v 1.4) | Thermo Fisher Scientific (Germany) | ||
| Recombinant DNAreagent | pKT0090 plasmid | PMID: 15197731 | Addgene:Plasmid #8714 | contains yEVenus |
| Recombinant DNAreagent | YEplac181 plasmid | PMID: 3073106 | Addgene:Plasmid #8628 | high copy plasmid |
| Recombinant DNAreagent | YCplac111 plasmid | PMID: 3073106 | Addgene:Plasmid #53249 | single copy plasmid |
| Recombinant DNAreagent | pRS315 plasmid | PMID: 2659436 | ATCC 77144 | low copy plasmid |
| Recombinant DNAreagent | pFA6a-TEV-6xGly-3xFlag-HphMX plasmid | Tim Formosa | Addgene:Plasmid #44083 | |
| Recombinant DNAreagent | pGAL-VHL-mCherry | PMID: 18756251 | galactose inducible VHL-mCherry | |
| Commercial assayor kit | µMACS GFP Isolation Kit | Miltenyi Biotec (Germany) | 130-091-125 | |
| Commercial assayor kit | μMACS DYKDDDDK Isolation Kit | Miltenyi Biotec (Germany) | 130-101-591 | *DYKDDDDK is also known as FLAG tag |
Additional files
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Supplementary file 1
Results of the Synthetic Genetic Array with yEVenus.
This file contains information of the Synthetic Genetic Array with yEVenus in single gene deletion backgrounds and was used to create the following figures/tables: Figure 1C, D, E and F, Figure 1—figure supplement 1 A–C, and Table 1.
- https://doi.org/10.7554/eLife.29845.010
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Supplementary file 2
Genetic interaction screen using alternative promoters.
The table contains fitness and genetic interaction data of chaperone-deficient mutants overexpressing yEVenus driven by alternative promoters. This file contains the dataset used to create Figure 3—figure supplement 1C.
- https://doi.org/10.7554/eLife.29845.011
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Supplementary file 3
Results of the GFP co-immunoprecipitation assay with yEVenus.
This file contains information of the GFP co-IP assay performed on the yEVenus overexpressing strain.
- https://doi.org/10.7554/eLife.29845.012
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Supplementary file 4
Results of the Sti1p co-immunoprecipitation assay.
This file contains information of the Sti1p co-IP assay performed on wild type and Sti1-3xFLAG strains under high and low protein burden, that is with high and low copy yEVenus plasmid, respectively. This file contains the dataset used to create Figure 3D.
- https://doi.org/10.7554/eLife.29845.013
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Supplementary file 5
All data used to create figures in the manuscript.
The file contains the datasets used to create specific manuscript figures: it contains the data for Figures 1A, 2A, B, C, D, 3A, B and C, Figure 3—figure supplement 1A,B.
- https://doi.org/10.7554/eLife.29845.014
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Transparent reporting form
- https://doi.org/10.7554/eLife.29845.015
