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

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.

Adapting to stress — chaperome networks in cancer

“…Thus, the network in type 2 tumours enables redundancy. Studies in yeast (BOX 2) and human cells indicate that other chaperome members may take over the workload of the disabled chaperome and retain a robust cellular network 30,35,104–106 . When HSP90 is impaired, alternative use of the HSP70 machinery is a logical solution, and several lines of evidence support this hypothesis.…”

“…The relationship between inhibition of chaperome network components and redundancy was addressed in a large-scale investigation of proteome changes following deletion of SSA1 and SSB1 (two HSP70 paralogues) in yeast that were grown under optimal conditions 30 . In addition to being highly abundant (both proteins are among the top 5% of yeast proteins by mass), Ssa1 and Ssb1 contain the most connections among all hub proteins, with 3,269 and 2,489 links to client proteins, respectively, and interact with >40 other chaperones 28 .…”

Absolute protein quantification of the yeast chaperome under conditions of heat shock

“…Published studies have also typically been limited to a subset of the chaperones/proteome, or focussed on transcriptional responses at the mRNA level. Proteomic studies have typically used SILAC approaches, pulse labelling with 35 S-methionine and semi-quantitative western blots to measure the proteome directly, whilst northern blots and DNA microarrays have inferred transcriptome changes [11][12][13][14][15][16][17][18]. Although we have previously quantified absolute protein abundance (copies per cell) for over 50 chaperones, the study was only performed under normal, chemostat growth conditions.…”

“…R. J. Mackenzie et alProteomics2016,16,[2128][2129][2130][2131][2132][2133][2134][2135][2136][2137][2138][2139][2140] …”

Transcriptomic analysis and driver mutant prioritization for differentially expressed genes from a Saccharomyces cerevisiae strain with high glucose tolerance generated by UV irradiation

“…18 Heat shock proteins in the cytoplasm or in the ER regulate protein folding and secretion, as molecular chaperones are important in the cellular stress response and are activated by various metabolic stresses. 19 Calnexin is an ER membrane protein that acts as a molecular chaperone and is associated with new glycoprotein synthesis and protein folding intermediates. 20,21 Under heat stress conditions, calnexin may interact with protein disulde isomerase (PDI), which is also a molecular chaperone in the ER, and may play a major role in the proteinfolding process.…”