RUI: Evolution of Phosphate Starvation Response in Yeast

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Intellectual merit. This project investigates how the phosphate signal transduction (PHO) pathway changes over evolutionary time in the Ascomycota lineage, with the goal of understanding how evolutionary transitions in this pathway alter growth in different niches. Using the yeasts Candida glabrata and Schizosaccharomyces pombe, these studies focus on the regulation of transcription, elucidation of the ancestral eukaryotic PHO pathway, and how phosphate starvation in all eukaryotes is sensed. Studies with C. glabrata will determine the binding specificity of the transcription factor Pho4. Pho4-regulated promoters in C. glabrata do not contain canonical CACGTG sequences and must utilize different sequences relative to Saccharomyces cerevisiae. The hypothesis that CgPho4 has multiple specificities for PHO promoters will be tested with in vivo (expression from promoter fragments) and in vitro (mobility shift) approaches. This project will capitalize on previous studies demonstrating the neofunctionalization of a C. glabrata acid phosphatase protein, Pmu2. Domains of Pmu2 will be combined with ancestral Pmu1 domains and phosphatase kinetics will be measured. The changes in protein sequence allowing for neofunctionalization will be identified and should be applicable to understanding how new functions evolve more generally. Studies with S. pombe will focus on the identification of Pho7 as a likely transcription factor mediating the phosphate starvation response in S. pombe. The hypothesis that Pho7 binds PHO promoters will be tested utilizing in vivo and in vitro approaches. Finally, the PHO pathway from Chlamydomonas (a green algae) and S. pombe will be genetically reconstituted in S. cerevisiae, allowing for the determination of whether common metabolites in all eukaryotes signal phosphate starvation. Phosphate starvation impacts important cell biological responses and altering the uptake of phosphate for bioremediation or altering the pathogenicity of bacteria and fungi would benefit society. The proposed work will advance our knowledge of transcription factor specificity, the process of neofunctionalization, the possible conservation of metabolic signals of phosphate starvation in all eukaryotes, and a deeper understanding of the evolutionary transitions required in pathways for speciation. Broader impacts. These studies are accessible to relatively new scientists such as undergraduate students and Master's level students. The experiments are parsed into small projects suitable for undergraduates, such as protein purification, enzymatic assays, and plasmid construction. Students will gain valuable experience working with genetics, strain construction, molecular biological techniques, sterile techniques, and biochemistry. Ownership of a task within the framework of a larger project is valuable for students' self-confidence and encourages future self-reliance. This work will support numerous undergraduate and Master's students at Villanova University who will go on to perform scientific research. Students from disadvantaged backgrounds are actively recruited and encouraged to stay in science by positive research experiences and strong mentorship.