VISUALIZING THE MACROMOLECULAR ORGANIZATION OF THYLACOID MEMBRANES USING CRYO-ET

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The photosynthetic membranes of chloroplasts, known as the thylakoids, are in some ways an ideal model system for studying many fundamental properties of biological membranes. These ways include their ease of isolation and purification, the fact that one can monitor general functional activities and the state of specific protein complexes in vivo, the availability of crystal structures of all major protein complexes within the thylakoids, and the availability of hundreds of mutants with altered thylakoid membrane proteins, lipids, developmental patterns and structure. The best resolution of protein complexes within the thylakoid membrane has thus far been achieved via freeze fracture, which has revealed the overall organization of the membrane. The objective of this project is to use frozen-hydrated specimens, combined with electron microscope tomography (ET), to investigate the macromolecular organization of the photosynthetic machinery within the thylakoid membranes in greater detail. With this methodology we not only have a chance to compare the macromolecular organization to the freeze fracture studies, but also to push the resolution to higher levels and investigate how the macromolecules interact with each other. This goal can be pursued both by looking at neighbors within and between thylakoid membranes, and by seeing how the crystal structures of these complexes or their components fit into the structural map of the thylakoid membrane developed with cryo-ET. We have obtained several single-axis tilt series of different preparations, including intact isolated and then cryo-sectioned spinach chloroplasts, isolated and plunge-frozen thylakoid membranes, and sections of intact Chlamydomonas cells. Preliminary results have enabled us to visualize the organization of the thylakoid stacks and the ATP synthase particles on the surface of the stroma thylakoids. We also found paracrystalline arrangements of not-yet-identified macromolecular complexes that resemble ordered complexes seen in freeze fracture images.