Deciphering the transcriptional response of saccharomyces cerevisiae to perturbations of lipid metabolism and graded endoplasmic reticulum stress

Abstract

Systems Biology combines experimental biology with mathematics and computational simulations to better describe biological phenomena that emerge from the interaction of different players. Extensive prior knowledge and experimental feasibility make the eukaryotic single-cell organism S. cerevisiae the preferred model organism for systems biology, while the strongly conserved features might enable conclusions for more complex organisms. In this thesis, a ‘Systems Biology’-approach was taken to better understand how S. cerevisiae coordinates different transcriptional and metabolic responses to adapt to two exemplary environmental changes, i.e. inositol depletion and low-level ER stress. Firstly, a quantitative model guided the construction of fast-folding, actively degraded reporter proteins, which were able to rapidly indicate specific transcriptional changes in single cells. Secondly, the developed reporter proteins, a fluorescent sphingolipid (SL) intermediate and classical molecular biology techniques were used to investigate the interaction of the signaling pathways, which enable S. cerevisiae to survive after inositol depletion, and to understand the role of SL metabolism during this process. The results highlighted the temporal order of transcription factors that follows the removal of inositol, i.e. first INO2/4, then HAC1 and lastly RLM1, and suggested that decreased SL biosynthesis is probably not responsible for the delayed disruption of ER homoeostasis but perturbs cell wall integrity after HAC1 activation. Thirdly, the adaptation to low ER stress was studied with a reporter protein for HAC1 and established fluorescent labels. The experimental insights then motivated a quantitative model for the adaptation to new environments, which lower the growth rate and change the inheritance of essential resources during cytokinesis. From the results, it emerged that ER stress mainly affects G1 duration in daughter cells and reduces the amount of ER content that is inherited by them. This lower inheritance probably contributed to the daughter-specific HAC1 activation. The analysis of the model implied that such a lower resource inheritance increases the daughter: mother ratio and probably lowers the resource demand of the population. Overall, the results supported the idea that transcriptional adaptation is primarily performed by daughter cells and is often a multi-step process. This work moreover lays the foundation to investigate transcriptional dynamics during other environmental changes and to further study the role of lipid metabolism for ER homeostasis. It also provided a mathematical model for the long-term impact of changes in the distribution of limiting resources.