The long range goal of this research is to gain insight into the selective transport of proteins from the cytoplasm into organelles with specialized metabolic functions. 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 polypeptide precursors, transported across a membrane barrier, and proteolytically processed. These studies will lead to an understanding of the general principles used to correctly route proteins in the cell. The import pathway of the light-harvesting chlorophyll a/b binding protein (LHCP) will be investigated. The LHCP gene has been inserted into a transcription vector to generate RNA and subsequently, labeled LHCP precursor (pLHCP) in vitro. Modifications in the pLHCP gene will be made using recombinant DNA methods to generate corresponding changes in the primary structure of the precursor. This approach provides a means of obtaining mutants in chloroplast assembly not yet achieved by conventional genetics. In a cell-free system, restructured proteins will be synthesized and incubated with isolated chloroplasts to determine how these changes affect binding to the organelle's envelope, transport across the membrane, intraorganelle routing and, of particular interest, precursor processing. A soluble processing enzyme that cleaves pLHCP has been identified and partially purified. The processing reaction will be optimized, and the determinants for pLHCP cleavage will be determined. The enzyme will be purified to homogeneity by column chromatographic methods, which have already been started, to establish its molecular composition and specificity. A gene coding for the enzyme will be isolated from a complementary DNA library and sequenced to study the structure of the polypeptide and its biosynthetic pathway. Processing enzymes play an essential role in organelle biogenesis; they are responsible for releasing the mature, functional proteins within the organelle. This research will have relevance to correcting metabolic disorders.
