Investigating the regulation of tumour associated lipogenesis using the model organism, Saccharomyces cerevisiae

Abstract

The highly conserved mitogen activated protein kinase (MAPK) high-osmolarity glycerol (HOG) pathway plays a vital role in the ability of Saccharomyces cerevisiae cells to respond to stress conditions, primarily hyperosmotic stress. In addition, there is also evidence that the HOG pathway is involved in lipid accumulation, which occurs in nitrogen depleted cells as they enter stationary phase. Understanding this lipogenic switch is important both for the biotechnology industry (for example biofuels) and understanding human disease, where cancer cells accumulate cellular lipid.

To investigate the role of the HOG1 pathway in neutral lipid accumulation, a Nile red assay was undertaken to measure lipid accumulation in stationary phase of genomic deletion strains of key HOG pathway proteins. Deletion of the majority of HOG components had no effect on lipid accumulation. However, the hog1Δ mutant showed a significant decrease in neutral lipids levels when compared to the wildtype. Hog1p is the key MAPK in the HOG pathway and this data suggests that it may also be a regulator of neutral lipid accumulation.

Hog1p is activated by phosphorylation, as such, a western blot analysis was performed to monitor dual phosphorylation of Hog1p during the lipogenic switch. This showed an increase in Hog1p phosphorylation between 9 – 10 hours of growth, which corresponds to the transitionary period between late exponential phase and early stationary phase and accumulation of neutral lipids.

Regulation of Hog1 is classically believed to be via dual phosphorylation by the MAPKK Pbs2. However, results from the Nile red assay of the pbs2Δ mutant, demonstrated that accumulation of neutral lipids is not affected in this strain when compared to the wildtype. Further, a hog1Δpbs2Δ double deletion showed no additive affect when compared to the individual deletion strains. This suggests that Pbs2p is not involved in the accumulation of neutral lipids, and, therefore, Hog1p is being activated by an alternative regulator. This was further investigated by monitoring Hog1p phosphorylation during the lipogenic switch in the absence of Pbs2p.

To investigate how Hog1 is regulating lipid accumulation, a number of potential neutral lipid downstream targets were investigated. One potential target is Dga1p, an enzyme that catalyses the terminal step of triacylglycerol formation in lipid production. Deletion of this protein results in reduction in neutral lipid levels as measured by the Nile red assay. A similar reduction in neutral lipid levels was observed in a hog1Δdga1Δ double deletion indicating the proteins may be involved in the same pathway. Dga1 being a target of Hog1 is supported by bioinformatics analysis, which identified four potential MAPK sites (S/T followed by P) within the protein.

This work indicates a potential novel role for Hog1p in regulating lipid accumulation in S. cerevisiae which may have wider implications for the study of lipid related diseases and for the biotechnological application of cellular lipids.