Investigating the heat shock response of the yeast proteome via quantitative proteomics

UoM administered thesis: Phd

  • Authors:
  • Rebecca Mackenzie

Abstract

Proteostasis, the regulation of protein abundance and function in cellular systems, underpins the ability of organisms to deal with environmental challenge such as heat shock stress. In this thesis, a quantitative study of this process at the molecular level has been undertaken using the model eukaryote S. cerevisiae to characterise the protein level response to heat shock. Although well studied, much of the previous work has focussed on changes at the mRNA levels or relative changes in protein expression. To address this, an absolute quantification strategy was developed utilising the QconCAT approach. A total of 10 recombinant QconCAT proteins were designed to target the 63 chaperones in S. cerevisiae, with up to 5 Q-peptides selected per chaperone where possible. Subsequently, absolute copy per cell values were determined for 49 of the 63 chaperones in S. cerevisiae under conditions of normal growth and heat shock (42 °C, 30 minutes). Chaperones that are known targets of the heat shock response activating transcription factor HSF1 are significantly upregulated in response to heat shock. Furthermore, this dataset has been extended towards proteome-wide quantification, for which SRM-normalised label free quantification values for 1644 proteins in both conditions were determined. Using these values and a high quality chaperone-client interaction dataset, progress has been made towards modelling the change in the protein volume and workload of each chaperone in response to heat shock. Interestingly, for the chaperone Ssb2, both its workload and absolute abundance were significantly upregulated in response to heat shock. However, across all chaperones, the relationship between protein volume, workload fold change and abundance fold change is minimal; further work is required to investigate this.

Details

Original languageEnglish
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Award date1 Aug 2017