9406540 Kindle Most chloroplast and mitochondrial proteins are encoded by nuclear genes, synthesized in the cytosol, and imported post translationally. Photosynthetic cells must be able to accurately sort proteins between mitochondria and chloroplasts. Targeting specificity is mediated by transit peptides which are present on cytosolic precursors and removed during or shortly after import into the organelle. Proteins that interact with the transit peptide during the import process presumably discriminate between chloroplast and mitochondrial precursors. This project seeks to apply the molecular and classical genetics of Chlamydomonas reinhardtii to questions concening chloroplast protein import. Stable nuclear mutants that are non-photosynthetic because they lack plastocyanin or the gamma-subunit of the chloroplast ATPase (CF1-gamma) have been isolated and can be transformed to photosynthetic competence using the corresponding wild-type structural gene. To define the region of the transit peptide that is required for chloroplast protein import in Chlamydomonas, the putative import domain will be subject to extensive deletion and site specific mutagenesis. The mutated gene will be reintroduced into the corresponding null mutant to determine whether it is able to complement the non-photosynthetic phenotype. The same mutations will be tested in a homologous in vitro chloroplast protein import assay. The results should give a detailed picture of the features of the transit peptide that are required for in vivo and in vitro import. A genetic selection will then be set up to identify extragenic supressors that are able to compensate for the transit peptide mutation. To minimize the number of revertants and transit peptide-linked suppressor mutations that are recovered, suppressors will be isolated using strains that carry two copies of the specfiic transit peptide mutation, one linked to the plastocyanin gene, the other linked to the CF1-gamma gene. Dep ending on the starting strain, accumulation of one or both proteins will be required to restore photosynthesis. In either case, accumulation of both gene products will be screened by immunoblot analysis. Suppressors that are unlinked to the original transit peptides will be of most interest since they may identify components of the protein import pathway that interact with the transit peptide. Chloroplasts will be prepared from candidate suppressors and tested in vitro for their ability to import precursors with the corresponding transit peptide mutation. The cloning of genes that are altered in such suppressor strains should define proteins that interact with the transit peptide during import. This work will provide insight into the requirements for transit peptide function and will identify proteins that interact with the transit peptide during chloroplast import. %%% With few exceptions, life on earth is completely dependent on photosynthesis, the process whereby light energy from the sun is used to convert atmospheric carbon dioxide plus water into sugar and oxygen. Chloroplasts are the subcellular site of photosynthesis in eukaryotic plants, and as such are critical to life on earth. Chloroplasts are semi-autonomous organelles within the plant cell, in which the photosynthetic machinery is separated from the rest of the cytoplasm by a set of two concentrically arranged cellular membranes. Since most of the proteins which comprise the photosynthetic machinery are synthesized in the cytoplasm, a mechanism is needed to bring those proteins across these two membranes into the chloroplast. The mechanism must be selective, since there are other membrane-bounded organelles in the cell, such as mitochondria and peroxisomes, which have different functions, and different protein contents in order to carry out those functions, and whose proteins are also imported from the cytosol. This project represents a very clever approach to two closely related question s: 1, what is the "signal" on certain proteins which destines those proteins specifically to the chloroplast?; and 2, what are the proteins in the chloroplast which are involved in the selective recognition of that signal and import of cytosolic proteins destined for the chloroplasts? If successful, the project will provide major advances in the understanding of chloroplast biogenesis and specificity of subcellular targeting. The information that will be obtained has obvious potential utility in agricultural biotechnology. ***
