RUI: Control Mechanisms for G1 Cyclin Subcellular Localization in Budding Yeast

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Distinct mechanisms for regulated movement of proteins into and out of the nucleus are critical when considering pathways that regulate basic cellular processes, such as cell division. This project focuses on the identification and characterization of genes important for the regulated movement of proteins into and out of the nucleus. The work will broadly impact our understanding of the cellular genes critical for nuclear localization signal (NLS) and nuclear export signal (NES) dependent protein movement, while clarifying the mechanism of regulated protein movement in the specialized case of budding yeast G1 type cyclins. Genes important for NLS and NES activity will be identified using Green Fluorescent Protein (GFP) based reporters and automated genome wide high throughput microscopy. Comparisons of distinct NLS bearing GFP reporters will be used to identify those genes generally required for this process as well as those genes specialized for specific NLS sequences. The physiological relevance of these genes to regulated protein movement will be addressed for the G1 cyclins. Localization, binding, and genetic assays will be carried out to elucidate the role of cellular factors in regulated movement of the G1 cyclin Cln3 and to measure the impact that this regulatedprotein movement has on cell cycle progression. Intellectual Merit: This work will lead to significant progress in the understanding of the mechanisms of regulated localization of G1 cyclins in budding yeast. This research will also identify genes important for signal sequence dependent nucleo-cytoplasmic movement that will be of value to the broader scientific community. The work represents a shift in focus for the PI and will introduce new technologies in the form of automated high throughput microscopy into the research laboratory. To facilitate this research, collaborations have been developed to gain access to expertise and resources not available at Rhodes College.Broader Impacts: This work will significantly impact the ability of the PI to give significant and engaged experiences to undergraduate researchers. The collaboration of Rhodes College and the University of Toronto, with the support of The Rockefeller University and the University of Virginia, provides a unique and invaluable opportunity for undergraduate researchers of various backgrounds to not only work together while using advanced technologies, but to make a significant impact on the basic biological understanding of nucleo-cytoplasmic protein transport. Individuals from groups generally underrepresented in US science will be actively recruited for this work, specifically through arrangements with LeMoyne Owen College in Memphis, TN (a private historically black small liberal arts college). This proposed work is especially well suited for undergraduate researchers. It utilizes clear, straight forward and well established methods to address basic and essential biological questions while also introducing the students to cutting edge technology in the arena of functional genomics. This project will fund undergraduate researchers during academic and summer months, increasing the culture of research in the Department of Biology at Rhodes College. Students and the PI will disseminate results from this study broadly to the scientific community through presentations at regional, national, and international meetings, as well as publication of data in highly regarded peer-reviewed journals.