Cell division and fusion are resource intensive activities, and thus the ability of the yeast cell to coordinate the availability of nutrients in its environment with reproduction is of great physiological importance. This project will examine the role that glucose availability plays in yeast mating by testing the hypothesis that glucose sensing and mating are interdependent. The glucose sensor and the pheromone receptor are the only G protein coupled receptors (GPCR) in yeast. The aims of this project include 1) assessing the effects of pheromone stimulation on the glucose sensor, 2) examining the direct physical interactions of the glucose sensing and pheromone receptors, and 3) identifying interactions between components of the signaling pathways downstream of these receptors.
Broader Impacts: This project, while enhancing our understanding of the relationship between nutrient sensing and mating in yeast, will impact undergraduate students and the science faculty at three primarily undergraduate institutions. Undergraduate students will be exposed to biochemical research during an intensive ten week summer project, with the opportunity to continue their research during the academic year. These students will be involved in the design, implementation, interpretation, and dissemination of the results of their own experiments in a community of their peers. This project represents an opportunity for the investigators to integrate their research into their teaching, thereby enhancing the scientific education of hundreds of students at the participating institutions. The students involved in this research will be primarily from a typically underserved population (including under-represented minorities) in rural Appalachia.
The overarching scientific goals of this research project were to demonstrate interdependence of a sugar-sensing pathway and the mating pathway in the baker’s yeast Saccharomyces cerevisiae and to determine how the two pathways interacted. This is of interest because S. cerevisiae is commonly used by scientists as a model organism so a full understanding of its metabolism is desirable, and because the types of pathways studied in this project are mediated by G protein-coupled receptors (GPCRs), a type of cell surface receptor that is responsible for activating many medically important biochemical pathways in humans. Through research supported by this award, we demonstrated that the low affinity glucose sensing pathway (Gpr1p-mediated) and the mating pathway are interdependent. Strong evidence was found to indicate that various components of the Gpr1p-mediated glucose sensing pathway are required for the mating pathway to function normally. When components of this glucose sensing pathway are absent from cells, mating and the processes that lead up to mating are inhibited (e.g. growth arrest, formation of mating projections, induction of genes associated with mating). These affects are more pronounced in yeast cells of one "gender" (MATa) than they are in the other (MATα). This indicates that, like many other yeast, S. cerevisiae has a mechanism to avoid entering the energetically costly process of mating if sufficient nutrients are not available in the environment. We systematically identified the proteins in the Gpr1p-mediated glucose sensing pathway that are required for normal activation of the mating pathway, and found that the amount of impact on the mating pathway varied at different levels of the pathway. This may indicate that there are at least two levels at which these pathways interact. In addition, we found evidence that the Gpr1p-mediated glucose sensing pathway is dependent on components of the mating pathway for normal function. When certain components of the mating pathway were removed, responses associated with sensing high levels of glucose in the environment (increased sensitivity to stress, increased glycogen accumulation) were inhibited. One point of contact between these pathways may be due to interaction between the G protein –coupled receptors that mediate these two pathways. Co-immunoprecipitation assays indicate that these receptors physically interact in cells overexpressing these receptors. Though this finding should be confirmed in cells at normal expression levels, the possibility that these two different GPCRs interact with functional consequences is intriguing. Such a system represents a model of interactions known to occur between many of the GPCRs in human cells, simplified by the fact that these are the only GPCRs in yeast cells. Mathematical models of the two pathways were created, and work to model the interactions between the pathways is ongoing. The primary broader impact of this award was to provide four faculty members at primarily undergraduate institutions and 29 undergraduates an opportunity to do research on a scale that is not normally feasible at a small teaching-intensive college. The funding supported 10 week summer undergraduate research experiences for 17 students, some of whom continued to work on the project during the academic year, as well as less intensive semester-long research projects for 12 undergraduate students. The students participating in the 10 week summer program consistently reported a significant improvement in their confidence in their laboratory skills and their understanding of the research process following their time in the lab. The students who participated in the summer program in years two and three of the award also reported an increase in their comfort with reading primary scientific literature after we instituted a formal journal club as part of their experience. Many of the students had the opportunity to present their research at regional and national meetings, where some won awards for their presentations. All of the students who participated in any aspect of the project graduated with, or are still seeking, a degree in science or mathematics. Students who worked on this project have gone on to research careers in industry and academia, medical school, veterinary school, and graduate school in various scientific disciplines. .
