Ubiquitin-mediated regulation of kinetochore assembly and function

Project Summary/Abstract An organism’s ability to grow, develop, and reproduce are some of the defining characteristics of life. Central to growth and reproduction of organisms ranging from single-celled baker’s yeast to humans is the ability of a cell to replicate its genome and accurately divide the genome into two daughter cells. Errors in the replication or division of the genome can result in genetic changes that cause disease or are lethal to the cell or organism. Segregation of the genome is accomplished through an intricate series of steps wherein spindle microtubules must successfully bind sister chromatids and pull one copy of each chromosome into each daughter cell. The kinetochore, a conserved megadalton protein complex, mediates microtubule attachment to chromosomes. Although prior work has successfully charted many kinetochore components, as well as key regulatory steps in kinetochore assembly and function, this process is still incompletely defined. The goal of this proposal is to understand how post-translational modifications, specifically ubiquitin, contribute to kinetochore assembly and function in the budding yeast, Saccharomyces cerevisiae. Using a combination of proteomics, yeast genetics, and biochemistry, I will generate a comprehensive map of kinetochore regulation by the Mub1/Ubr2 E3 ubiquitin ligase complex (Aim 1) and investigate how a large family of E3 ubiquitin ligases, the cullin-RING ligases, regulates kinetochore function (Aim 2). Combined, these approaches will allow me to address how ubiquitylation influences kinetochore function and generate new knowledge surrounding kinetochore regulation. Given the highly conserved nature of the kinetochore, this work will likely identify principles of kinetochore regulation that apply to multiple organisms. Understanding these principles could provide insight into the cellular adaptations that occur in response to pathological changes in chromosome number (aneuploidy), a common feature of cancer cells. The training facilitated by this fellowship, along with my previous research experiences, will allow me to develop the skills necessary to become an independent academic investigator, with the long-term goal of establishing a research program that uses yeast and mammalian systems to study mechanisms of kinetochore regulation.