When conditions change, unicellular organisms rewire their metabolism to sustain cell maintenance and cellular growth. Such rewiring may be understood as resource re-allocation under cellular constraints. Eukaryal cells contain metabolically active organelles such as mitochondria, competing for cytosolic space and resources, and the nature of the relevant cellular constraints remain to be determined for such cells. Here, we present a comprehensive metabolic model of the yeast cell, based on its full metabolic reaction network extended with protein synthesis and degradation reactions. The model predicts metabolic fluxes and corresponding protein expression by constraining compartment-specific protein pools and maximising growth rate. Comparing model predictions with quantitative experimental data suggests that under glucose limitation, a mitochondrial constraint limits growth at the onset of ethanol formation—known as the Crabtree effect. Under sugar excess, however, a constraint on total cytosolic volume dictates overflow metabolism. Our comprehensive model thus identifies condition-dependent and compartment-specific constraints that can explain metabolic strategies and protein expression profiles from growth rate optimisation, providing a framework to understand metabolic adaptation in eukaryal cells.

When conditions change, unicellular organisms rewire their metabolism to sustain cell maintenance and cellular growth. Such rewiring may be understood as resource re-allocation under cellular constraints. Eukaryal cells contain metabolically active organelles such as mitochondria, competing for cytosolic space and resources, and the nature of the relevant cellular constraints remain to be determined for such cells. Here we developed a comprehensive metabolic model of the yeast cell, based on its full metabolic reaction network extended with protein synthesis and degradation reactions (16304 reactions in total). The model predicts metabolic fluxes and corresponding protein expression by constraining compartment-specific protein pools and maximising growth rate. Comparing model predictions with quantitative experimental data revealed that under glucose limitation, a mitochondrial constraint limits growth at the onset of ethanol formation - known as the Crabtree effect. Under sugar excess, however, a constraint on total cytosolic volume dictates overflow metabolism. Our comprehensive model thus identifies condition-dependent and compartment-specific constraints that can explain metabolic strategies and protein expression profiles from growth rate optimization, providing a framework to understand metabolic adaptation in eukaryal cells.

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Transcription factors (TFs) consist of a DNA binding and an activation domain (AD) that are considered to be independent and exchangeable modules. However, recent studies conclude that also the physico-chemical properties of the AD can control TF assembly at chromatin via driving a phase separation into “transcriptional condensates”. Here, we dissected the mechanism of transcription activation at a reporter gene array with real-time single-cell fluorescence microscopy readouts. Our comparison of different synthetic TFs reveals that the phase separation propensity of the AD correlates with high transcription activation capacity by increasing binding site occupancy, residence time and the recruitment of co-activators. However, we find that the actual formation of phase separated TF liquid-like droplets has a neutral or inhibitory effect on transcription induction. Thus, our study suggests that the ability of a TF to phase separate reflects the functionally important property of the AD to establish multivalent interactions but does not by itself enhance transcription.