Feedback regulation mechanisms and the rate-determining step of the yeast MAP kinase pathway

Abstract

Three-tier module of the MAP kinase pathway is a classical type of signal transduction system well-known for regulating cell functions in response to diverse external stimuli. MAP kinase pathways exist in all types of eukaryotes with its vast number of components and connections to control cells fate. For such complex system to function properly within the seemingly chaotic cellular environment, MAP kinase pathway must be tightly regulated for a reliable signal transmission. Many of the key components, participating proteins, and their interacting network have been identified. However, we know far less about the core structure (connections) that controls the three-tier module. MAP kinase pathway is tightly regulated for a reliable signal transmission and ultimately decision making of the cell. In this study, we explored the rate-determining step and the core control circuit by combining experimental results, mathematical modeling and comparison between the two. To experimentally identify the rate-determining step of the MAP kinase pathway, we used kinases from the yeast mating pathway as a model. We manipulated abundances of three key players of the yeast mating pathway. The three kinases form the three-tier that is sequentially activated and therefore essential to the signal transmission of the mating pathway. We modified protein abundance by under-expression and over-expression of each kinase and observed how the overall signal transduction is affected. Previously, reducing protein expression is mainly limited by the difficulties associated with controlling the reduction level and in some cases the initial endogenous abundance is too low. For the under-expression to be useful as an experimental tool, repeatability and stability of reduced expression is important. We found that cis-elements in programmed 1 ribosomal frameshifting (1RFS) of beet western yellow virus (BWYV) could be utilized to reduced protein expression in Saccharomyces cerevisiae. The two main advantages of using 1RFS are adjustable reduction rates and ease of use. Programmed 1RFS was used for under-expression whereas over-expression was achieved with stronger promoter and high-copy plasmids. Three kinases were subjected to six variations of abundance differing from their endogenous expression levels. The results showed that signal output decreased as Ste11 abundance decreased and the output increased as the abundance of Ste11 increased, identifying ste11 as the rate-determining component. We have developed a novel mathematical model of the yeast mating pathway to investigate how ste11, MAPKKK, alone affects the overall outcome and to find the rate-determining step and the controlling regulation mechanisms of the yeast decision making system. We constructed a three component model with all possible connections which comes to a total of 2187 structures. 2187 structures went through multiple analyses including elimination process, fitness analysis, and synergy analysis to yield a core structure that represents the yeast mating pathway. The optimal structure revealed that at least two negative feedbacks and a positive feedback are needed as a regulation mechanism to explain the rate-determining effects of ste11 and robustness of Ste7 and Fus3. Positive feedback can be explained with increased output strength in a constitutively active ste11 mimicking amplification taking place between ste11 and ste7. Addition to multiple negative regulations already known from previous studies, we identify new negative regulations on ste7 by Ppq1 which seems to be activated by Fus3. Combination of these regulations facilitates the three component signal transduction system for a controlled and robust response. Cells also face randomness of life with equally complex orchestration of internal stochasticity and controlled stability. Cell-to-cell variations, even in an isogenic population are inevitable and increasing evidence suggests that cells can strategically limit the noise but also benefit from the variation caused by the noise. However, origins, characteristics and significance of these variations are partially known and remain controversial. Here, we track MAP kinase signaling pathway responses of yeast single cells and quantitatively analyze each response profiles to find the existence of response profile stochasticity (RPS). We find that response profiles of isogenic yeast populations are distinct enough to be divided into groups that differed in response speed and duration. Interestingly, RPS is an inherent trait that increases diversity as an individual by allowing different responses every time while maintaining a consistent and robust average response as a population. Our findings indicate that yeast cells increase survival chance at both population and individual levels with RPS originating from the MAP kinase signaling pathway by diversifying under identical genetic and environment backgrounds.