Plant cells are unique, among eukaryotic cells, in that they contain two organelles, the mitochondria and the chloroplasts, thought to be derived from prokaryotic endosymbionts in an ancestral organism. Most of the proteins in these organelles are encoded in the nuclear genome and are synthesized in the cytoplasm. These proteins are produced as precursors, with N-terminal extensions of their polypeptide chains known as transit peptides. These precursors bind to receptors on the two organelles and are transported inside, where the transit peptides are proteolytically removed. Although the two import systems are similar in many respects, the transit peptides seem capable of conferring complete targetting specificity, in vivo, if they are fused to a "passenger protein." Analysis of the structure of the two types of transit peptides have revealed differences related to proposed domain structures. The aim of this project is to determine what structural properties of the transit peptides confer targetting specificity, distinguishing them, if possible, from properties required simply for import into the isolated organelles. In order to do this, artificial transit peptide / passenger protein fusions will be prepared and assayed for their ability to be imported into isolated plant organelles and in transgenic tobacco plants. The prototype transit peptide sequences will be derived from a chloroplast pre-protein of pea and from a mitochondrial pre-protein from Arabidopsis. The project addresses a very important problem in cell biology, that is, intracellular compartmentation. In order for cells to function properly, it is necessary that the macromolecules of the cell be organized in such a way as to facilitate cellular processes. This compartmentation is achieved through molecular specificity and recognition at the cellular level. In the case of the two organelles under study in this project, mitochondria and chloroplasts, the macromolecular components of each consist of a mixture of nuclear gene encoded and organelle-specific gene encoded products. The two organelles have striking similarities: they contain electron-transporting protein complexes and catalyze bioenergetic reactions; they contain specific DNA, RNA, and functional ribosomes; the internal spaces are separated from the cytoplasm by a double set of membranes; and the mechanisms by which nuclear-encoded proteins enter the organelles seem very similar. However, nuclear chloroplast proteins do not find their way into mitochondria, and nuclear mitochondrial proteins do not find their way into chloroplasts. The molecular mechanism which confers this critical organelle specificity to the import process is unknown. This set of experiments is likely to shed light on this important question.
