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Quantitative proteomics and network analysis of SSA1 and SSB1 deletion mutants reveals robustness of chaperone HSP70 network in Saccharomyces cerevisiae

View Article: PubMed Central - PubMed

ABSTRACT

Molecular chaperones play an important role in protein homeostasis and the cellular response to stress. In particular, the HSP70 chaperones in yeast mediate a large volume of protein folding through transient associations with their substrates. This chaperone interaction network can be disturbed by various perturbations, such as environmental stress or a gene deletion. Here, we consider deletions of two major chaperone proteins, SSA1 and SSB1, from the chaperone network in Sacchromyces cerevisiae. We employ a SILAC‐based approach to examine changes in global and local protein abundance and rationalise our results via network analysis and graph theoretical approaches. Although the deletions result in an overall increase in intracellular protein content, correlated with an increase in cell size, this is not matched by substantial changes in individual protein concentrations. Despite the phenotypic robustness to deletion of these major hub proteins, it cannot be simply explained by the presence of paralogues. Instead, network analysis and a theoretical consideration of folding workload suggest that the robustness to perturbation is a product of the overall network structure. This highlights how quantitative proteomics and systems modelling can be used to rationalise emergent network properties, and how the HSP70 system can accommodate the loss of major hubs.

No MeSH data available.


Volcano plots for the two Hsp70 yeast mutant proteomes compared to wild‐type yeast. Volcano plots show the normalised log2‐fold change and calculated ANOVA p‐value (–log10) for the SSA1, SSB1, chaperone mutants. Solid red line is at p value = 0.05 and points above it represent significantly changing proteins. The blue points represent proteins with FDR adjusted p‐value < 0.05 and dashed horizontal lines are positioned at log2 fold changes of –1 and 1, corresponding to two‐fold down/upregulation. Very few proteins exceed these cutoffs with statistical significance.
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pmic8085-fig-0005: Volcano plots for the two Hsp70 yeast mutant proteomes compared to wild‐type yeast. Volcano plots show the normalised log2‐fold change and calculated ANOVA p‐value (–log10) for the SSA1, SSB1, chaperone mutants. Solid red line is at p value = 0.05 and points above it represent significantly changing proteins. The blue points represent proteins with FDR adjusted p‐value < 0.05 and dashed horizontal lines are positioned at log2 fold changes of –1 and 1, corresponding to two‐fold down/upregulation. Very few proteins exceed these cutoffs with statistical significance.

Mentions: After linear regression normalisation, ANOVA statistics were calculated to determine the differentially regulated proteins between mutant and wild‐type yeast. Volcano plots showing the normalised log2 protein ratio and associated statistical significance measure (–log10 p value) for the two chaperone mutants are shown in Fig. 5. A full list of proteins along with log2(L/H ratios) pre‐ and postnormalisation, p values and adjusted p‐values and raw intensities from MaxQuant is provided in Supporting Information Table 1 in Supplementary Data. Based on replicate measurements and a standard ANOVA test, 103 ‘down’ and 86 ‘up’‐regulated proteins were identified as significantly changing in the ΔSSB1 mutant and 15 ‘down’, 18 ‘up’ in ΔSSA1 (p < 0.05). To control the FDR, Benjamini–Hochberg multiple testing correction 40 was applied, resulting in 27 down‐regulated proteins and 11 as up‐regulated for ΔSSB1 at an adjusted p‐value threshold of 0.05. No proteins passed this threshold in the ΔSSA1 mutant.


Quantitative proteomics and network analysis of SSA1 and SSB1 deletion mutants reveals robustness of chaperone HSP70 network in Saccharomyces cerevisiae
Volcano plots for the two Hsp70 yeast mutant proteomes compared to wild‐type yeast. Volcano plots show the normalised log2‐fold change and calculated ANOVA p‐value (–log10) for the SSA1, SSB1, chaperone mutants. Solid red line is at p value = 0.05 and points above it represent significantly changing proteins. The blue points represent proteins with FDR adjusted p‐value < 0.05 and dashed horizontal lines are positioned at log2 fold changes of –1 and 1, corresponding to two‐fold down/upregulation. Very few proteins exceed these cutoffs with statistical significance.
© Copyright Policy - creativeCommonsBy-nc
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4979674&req=5

pmic8085-fig-0005: Volcano plots for the two Hsp70 yeast mutant proteomes compared to wild‐type yeast. Volcano plots show the normalised log2‐fold change and calculated ANOVA p‐value (–log10) for the SSA1, SSB1, chaperone mutants. Solid red line is at p value = 0.05 and points above it represent significantly changing proteins. The blue points represent proteins with FDR adjusted p‐value < 0.05 and dashed horizontal lines are positioned at log2 fold changes of –1 and 1, corresponding to two‐fold down/upregulation. Very few proteins exceed these cutoffs with statistical significance.
Mentions: After linear regression normalisation, ANOVA statistics were calculated to determine the differentially regulated proteins between mutant and wild‐type yeast. Volcano plots showing the normalised log2 protein ratio and associated statistical significance measure (–log10 p value) for the two chaperone mutants are shown in Fig. 5. A full list of proteins along with log2(L/H ratios) pre‐ and postnormalisation, p values and adjusted p‐values and raw intensities from MaxQuant is provided in Supporting Information Table 1 in Supplementary Data. Based on replicate measurements and a standard ANOVA test, 103 ‘down’ and 86 ‘up’‐regulated proteins were identified as significantly changing in the ΔSSB1 mutant and 15 ‘down’, 18 ‘up’ in ΔSSA1 (p < 0.05). To control the FDR, Benjamini–Hochberg multiple testing correction 40 was applied, resulting in 27 down‐regulated proteins and 11 as up‐regulated for ΔSSB1 at an adjusted p‐value threshold of 0.05. No proteins passed this threshold in the ΔSSA1 mutant.

View Article: PubMed Central - PubMed

ABSTRACT

Molecular chaperones play an important role in protein homeostasis and the cellular response to stress. In particular, the HSP70 chaperones in yeast mediate a large volume of protein folding through transient associations with their substrates. This chaperone interaction network can be disturbed by various perturbations, such as environmental stress or a gene deletion. Here, we consider deletions of two major chaperone proteins, SSA1 and SSB1, from the chaperone network in Sacchromyces cerevisiae. We employ a SILAC&#8208;based approach to examine changes in global and local protein abundance and rationalise our results via network analysis and graph theoretical approaches. Although the deletions result in an overall increase in intracellular protein content, correlated with an increase in cell size, this is not matched by substantial changes in individual protein concentrations. Despite the phenotypic robustness to deletion of these major hub proteins, it cannot be simply explained by the presence of paralogues. Instead, network analysis and a theoretical consideration of folding workload suggest that the robustness to perturbation is a product of the overall network structure. This highlights how quantitative proteomics and systems modelling can be used to rationalise emergent network properties, and how the HSP70 system can accommodate the loss of major hubs.

No MeSH data available.