Fluorescence-Based Investigation of the Structure and Functional Dynamics of the Mitochondrial Protein Import Machinery

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Intellectual MeritMitochondria are organelles that play central roles in fundamental cellular processes including energy metabolism and programmed cell death. There are roughly 1000 (yeast) to 1500 (man) proteins resident within mitochondria, nearly all of which are synthesized outside the organelle and then trafficked to the mitochondrion by molecular complexes in the mitochondrial membrane. Our knowledge of the operation of these multi-component transport complexes has grown immensely in recent years. However, because these complexes are bound within the mitochondrial membranes, they are recalcitrant to many traditional techniques that are used to study protein structure and function. Hence, fundamental questions regarding the activity of these essential complexes have remained unaddressed. This project will employ a novel fluorescence-based approach to study the function and biogenesis of the central mitochondrial protein transporter, the TIM23 complex. Variants of the core channel-forming subunit (Tim23) labeled with small fluorescent probes at specific sites will be analyzed both in the context of native membranes (mitochondria isolated from yeast) and in reconstituted model membrane systems. Insights into the structural dynamics of the complex will be obtained using both environment-sensitive fluorescent probes and fluorescence energy transfer. Moreover, because labeled complexes are functionally active, this approach allows the analysis of structural changes as they occur in real time in response to different effectors. This innovative approach will not only enhance our understanding of how proteins are transported across biological membranes but, more generally, it will provide insights into the dynamics and interactions of membrane proteins and pave the way for future fluorescence-based investigations of other membrane protein complexes. Broader Impacts This research program will provide the basis for multiple educational and outreach components. Research training will include two teams of graduate and undergraduate students, each focusing on specific objectives. Collaborative interactions with cell biologists, molecular geneticists and biophysicists will foster a multidisciplinary training program. Educational activities of the project will include incorporation of research findings into two upper-division courses taught by the PI: "The Structure and Function of Biological Membranes" and "Principles of Cellular Bioenergetics". In addition, research will be integrated into a series of laboratory-based modular summer courses on the principles of fluorescence spectroscopy, giving undergraduates exposure to cutting-edge instrumentation and research on complex molecular systems. The scientific outreach component will include sponsorship of students from underrepresented groups through a Summer Research Program for Minority Undergraduates. In this program, highly motivated undergraduates from regional colleges attend a ten-week summer research course in which they are trained in multiple techniques and given the opportunity to present their findings at regional scientific meetings.