Mitochondria 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.
Mitochondria are organelles within our cells that are responsible for not only producing the majority of our metabolic energy, but also for synthesizing different macromolecules, regulating cellular homeostasis, and organizing programmed cell death. The research conducted under this NSF-sponsored program addressed the how particular membrane-bound machineries of the mitochondrion operate and are regulated to perform their myriad tasks (Figure 1). This research program utilized many technically innovative experimental approaches to study membrane proteins and complexes. First, we used a high-resolution fluorescence mapping strategy whereby environment-sensitive reporter probes are incorporated into specific sites on protein subunits (Figure 2A). Using advanced analytical fluorescence spectroscopy techniques, this approach allows us to directly analyze localized structural dynamics of membrane proteins as they occur in real time. Second, we employed a range of specially designed model membrane systems that allow us to use a range of biochemical and biophysical techniques to study membrane proteins and complexes in a well-defined lipid environment (Figure 2B). Combined with a range of cross-disciplinary techniques, including site-specific crosslinking, computer molecular dynamics simulations, mass spectrometry, and small angle x-ray scattering, this research program gave unprecedented insights into how mitochondrial protein complexes function at the molecular level. First, we investigated the structural dynamics of the TIM23 protein transport complex of the mitochondrial inner membrane. This complex mediates the transport and sorting of most of the nuclear-encoded proteins that reside within the mitochondrion, so its operation is of fundamental importance to the biogenesis of the organelle. Using our site-specific fluorescence mapping and crosslinking approaches in active mitochondria, we discovered how Tim23, the central voltage-gated channel forming subunit, changes its structure in response to alterations in the transmembrane electric field (Figure 3A). This work provided novel insights into the nature of electromechanical coupling in membrane proteins (i.e., how proteins transduce electrochemical gradients into work to drive useful processes) and, more specifically, how this protein transport channel may undergo gating during polypeptide transport. Further, using a host of model membrane reconstructions, we analyzed protein-protein and protein-lipid interactions within this complex. This work revealed that interactions among the subunits are lipid-mediated, specifically promoted by cardiolipin, the signature phospholipid of the mitochondrion (Figure 3B). Taken together, this research has strongly advanced our understanding of the molecular mechanisms that underpin the operation of this complex. Second, we analyzed the lipid dependence of the structure and function of complex II, the succinate:ubiquinone oxidoreductase (Figure 4A). This four-subunit respiratory complex mediates the reduction of succinate, an intermediate of the citric acid cycle, and transfers the electrons to reduce the quinone pool. Because this complex acts as a link between two fundamental energy transducing processes – the TCA cycle and the electron transport chain – it is likely to play many as yet undiscovered roles in the regulation of cellular energetics. Using a reductionist approach, we provided the first evidence that cardiolipin in required for the holoenzyme stability of complex II (stable interaction between the membrane and soluble heterodimers) and for the redox activity (Figure 4B). This enzyme is not only fundamental to bioenergetics, but its dysfunction is implicated in a number of mitochondrial diseases and heritable forms of cancer. Our progress on understanding the lipid-dependence of its operation has advanced our understanding of this complex in human health and disease. Finally, we have investigated the lipid-dependent activity of complex IV, cytochrome c oxidase, using a host of model membrane systems (Figure 5A). This work has focused on the activity of this respiratory complex in the presence of cardiolipin variants. Understanding the coupling between cardiolipin physiochemical properties and respiratory complex function is relevant to lipid synthesis and heritable remodeling defects (Figure 5B). This work has advanced our understanding of the role of key lipids in membrane complex activation (Figure 5C). It has also given significant insights into the chemical properties of cardiolpin, including its proton dissociation behavior and divalent cation binding activity, relevant to mitochondrial surface charge and calcium buffering properties. The broad impacts of this research program include our technical advances in the development of tools for cotranslational polypeptide labeling and novel model membrane systems for analyzing membrane proteins, all of which we have shared with the scientific community through technical publications and direct collaboration. Moreover, under NSF-sponsorship, our group has engaged in many scientific outreach programs with the public. This has included our Biology Summer Institute program serving as professional development for high school teachers in conjunction with the University of Connecticut ECE Program; the Advanced Research Mentorship program, in which high school students conduct year-long research activities under the guidance of a university instructor; and our Summer Research Program for Minority Undergraduates, in which students from groups underrepresented in the sciences have the opportunity to conduct and present research projects.
