Robustness analysis of eukaryotic signal transduction with emphasis on the HOG pathway in Saccharomyces cerevisiae

Abstract: Fundamental to life, in particular for unicellular microorganisms that generally are subjected to rapidly changing environmental parameters, is the need to monitor and integrate environmental stimuli with the internal cell status. The sensing of stimuli are typically initiated at the plasma membrane and then transmitted by means of intracellular signalling networks to elicit proper cellular responses. To separate genuine signals from fluctuations in components levels of the main players in transmission of these signals, signalling networks must be robust to such fluctuations. Here we investigate robustness of signalling pathways by systematic perturbation of the expression of these signalling components. To this mean, a plasmid-based overexpression methodology named “genetic tug-of-war” or gTOW was used. With this method, the upper permissible limit of gene copy number and hence expression level of a target gene can be evaluated. It differs from more commonly used inducible promoter systems such as the pGAL1-driven in that the regulatory regions (promoter and terminator) of a target gene are maintained on the plasmid. This feature allows a regulated gene control but at the same time causes an increase in gene expression proportional to the increase in plasmid copy number. Here we focus on the well-characterized High Osmolarity Glycerol (HOG) pathway in the yeast Saccharomyces cerevisiae that is essential for survival under conditions of high external osmolarity. We observe a high frequency of fragile nodes within the HOG pathway and that this fragility disperses over all main players in signal transduction as well as both positive and negative pathway regulators. This is exemplified by the finding that the most fragile nodes were the response regulator SSK1, the MAPK PBS2 (positive pathway regulator) and the phosphatase PTC2 (negative pathway regulator). Furthermore, the fragility patterns are largely independent of the overall pathway activation status (by varying the input stimuli). In addition, we found that toxicity from overexpression seem to be linked, at least in part, to pathway hyperactivation, demonstrated by the fact that toxicity could be suppressed by deletion of upstream or downstream pathway components. This gTOW imposed robustness analysis will be further extended to other signalling pathways in yeast with the ultimate goal of a complete robustness analysis of the entire signal transduction network in Saccharomyces cerevisiae.