Nuclear Function of a Membrane Ubiquitin Ligase

University Of North Carolina At Chapel Hill, Chapel Hill NC

Investigators

Abstract

Intellectual merit. Most cellular events are controlled by precise regulation of protein expression. Termination of protein expression is commonly achieved by moving proteins into cellular recycling centers, where they are broken down into amino acids that can be reutilized in new protein synthesis. Entry into these recycling centers is tightly regulated. Proteins that are turned-over inside proteasomes, barrel-shaped recycling centers in the cytoplasm, need a special modification that serves as a key or signal for entry. This "key" is a small sequence of amino acids, named ubiquitin, which is linked or ligated to proteins by a class of regulatory proteins known as ubiquitin ligases. Ubiquitin ligases thus control protein expression by their ability to recognize and tag other proteins for destruction. Ubiquitin ligases have multiple substrates and thus commonly impact multiple key cellular pathways, including regulation of the cell cycle, programmed cell death, and the integration of signaling pathways during development. Uncovering the function of ubiquitin ligases is essential to our understanding of all cellular processes. Most ubiquitin ligases are soluble proteins that ubiquitinate cytoplasmic proteins. This research will focus on identifying the proteins tagged for turnover by a specific ubiquitin ligase, an endosomal membrane protein that is stabilized and moves to the inner nuclear membrane when cell signaling pathways under the outer cell membrane are activated. To identify proteins tagged by this ligase, experiments will compare the protein composition of purified inner nuclear membrane when the ligase is active and when its expression is knocked down. Proteins that escape ubiquitin-mediated degradation and thus increase in quantity in the inner nuclear membrane fraction when expression of the ligase is knocked down will be identified by mass spectrometry. Identification of ligase substrates correlated with specific nuclear functions will indicate a role for the endosomal ubiquitin ligase in nuclear regulation. Broader impacts: These studies will serve as a vehicle for educating undergraduates, graduate students, and postdoctoral fellows, including members of under-represented groups. The project is based on observations initially obtained by a graduate student. The studies were confirmed and expanded by a postdoctoral fellow. The principal investigator has and will continue to train undergraduate chemistry and biology majors, who either elect research for course credit or spend the summer in the lab with REU grant support. The undergraduates attend lab meetings, have their own projects, and are included on publications. All trainees not only master new techniques but, more importantly, learn to analyze data, to propose creative solutions to problems, and to summarize and present data in a professional format. Mentoring undergraduates is excellent training for the graduate students and postdocs in the lab, teaching them to teach, to organize and schedule, and to encourage others.