PROBING THE SEC-LIKE THYLAKOID PROTEIN IMPORT PATHWAY

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Signal recognition, signal transduction, protein translocation across membranes, energy transduction and protein folding are major areas of investigation in biology since our understanding of infectious diseases and cell growth and death is dependent upon a comprehension of these phenomena. Contributions to all of these fields of research can be made by studying protein import into chloroplast thylakoids in real time. Recently, it has become clear that one of the thylakoid protein import pathways is similar to the prokarotic (Escherichia coli) protein secretion pathway which involves at least seven "Sec" proteins. This Sec like thylakoid import pathway is thought to be a direct result of the endosymbiotic origin of chloroplasts from an ancestral cyanobacterium. In the research proposed here, the interactions of a precursor protein with the Sec-like thylakoid import apparatus will be investigated with fluorescent, spin-labeled, and photoactive probes attached to ribosome-bound precursors of different lengths. In addition, the import of a full-length, probe-labeled precursor will be initiated by photoproduction of a transmembrane proton motive force and the import process will be monitored in real time (stopped-flow regime) using fluorescence and/or electron paramagnetic resonance spectroscopy. Spectral dat gathered from the interrupted import experiments (ribosome- bound precursors) are expected to aid in interpretation of the real time measurements. These investigations of thylakoid protein import will directly influence our understanding of prokaryotic protein secretion.