The long range goal of this research is to gain insight into the selective transport of proteins from the cytoplasm into specialized organelles. This work will focus on the import of proteins into the chloroplast. The chloroplast is one of the two energy producing organelles in eucaryotes. Like the mitochondrion, most of its proteins are encoded by the nuclear genome, synthesized as precursors, and transported across a membrane barrier before assembly. The current grant will investigate the signals used to selectively target proteins to one organelle, and will determine how the organelle-specific proteins are localized in the correct compartment after import. Isolated wheat genes coding for two important chloroplast proteins, the chlorophyll a/b binding protein of the light harvesting complex (cabLHC) and the small subunit of ribulose 1,5-bisphosphate carboxylase (rbcS), will be modified by recombinant DNA methods, i.e. site-directed mutagenesis. In a cell-free system, restructured proteins will be synthesized and incubated with isolated chloroplasts to investigate the effects on import. This approach provides a means of obtaining mutants in chloroplast assembly not yet achieved by conventional genetics. These studies will lead to an understanding of the general principles used to route proteins in the cell. Eventually it should be possible to target any modified protein to a specific cytoplasmic organelle. This research may have relevance to correcting metabolic disorders.
