Assembly of photosystem I in thylakoid membranes

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Photosystem I (PSI) is one of the largest and most intricate macromolecular machineries in nature. While the structure of PSI has been resolved in great detail, very little is known about how the complex is put together from more than a dozen protein subunits and more than 200 pigments and other co-factors. In the current funding period, we have (i) developed a new biochemical approach that has led to the discovery of two intermediate complexes in PSI assembly of seed plants, (ii) set up in vivo tagging and affinity purification approaches that have yielded a number of interesting candidate proteins for new components of the PSI-assembling machinery, and (iii) designed a new time-resolved system to study the sequence of events in PSI assembly of the green algal model Chlamydomonas reinhardtii. We now plan to build upon this work and (i) functionally characterize the novel putative PSI assembly factors we have isolated, (ii) determine their precise role in the PSI assembly process, and (iii) use the established inducible PSI knock-down and recovery system in Chlamydomonas to biochemically resolve individual steps in PSI assembly. The ultimate goals of the research proposed here are to elucidate the molecular structure of the PSI-assembling machinery and determine the functions of its components, establish an assembly map for PSI that provides a complete picture of the spatial and temporal sequence of events in the assembly process, and elucidate the evolution of the assembly machinery in relation to the evolution of PSI structure and thylakoid morphology.