This proposal seeks to define biochemically information present in a nuclear encoded protein that specifies its correct placement within the chloroplast. Genetic screens using heterotrophically grown plants will also be developed to identify cellular components that interact with this information. The light harvesting chlorophyll a/b binding protein pLHCP is coded for in the nucleus and post-translationally transported to the chloroplast as a precursor, where it assembles into the thylakoid as an integral membrane complex of pigment and chlorophyll. Isolated chloroplasts and radiolabelled in vitro mutagenized pLHCP will be used to study the import process and the suborganellar localization. A carboxy-terminus membrane spanning sequence and a charged carboxy-terminal are required for correct membrane integration. A series of specific mutations and genes fusions with soluble proteins will be used to characterize these regions and determine whether they act as independent signals or confer a specific folding pattern on the entire polypeptide. The surface of the chloroplast will be analyzed biochemically through the isolation of proteins that can bind precursors. These receptors will be isolated by affinity chromatography using solid supports and crosslinked precursor sequences. In addition antibodies to surface exposed chloroplast proteins will be used to isolate cDNA clones from an expression library. A genetic screen using a fusion of pLHCP and a selectable marker protein normally not found in a plant will be developed for the isolation of nuclear loci that regulate the transport of precursors to the chloroplast. The screen will take advantage of the heterotrophic capabilities of photosynthetically impaired higher plants.
| Auchincloss, A H; Alexander, A; Kohorn, B D (1992) Requirement for three membrane-spanning alpha-helices in the post-translational insertion of a thylakoid membrane protein. J Biol Chem 267:10439-46 |
| Kohorn, B D; Lane, S; Smith, T A (1992) An Arabidopsis serine/threonine kinase homologue with an epidermal growth factor repeat selected in yeast for its specificity for a thylakoid membrane protein. Proc Natl Acad Sci U S A 89:10989-92 |
| Kohorn, B D; Auchincloss, A H (1991) Integration of a chlorophyll-binding protein into Escherichia coli membranes in the absence of chlorophyll. J Biol Chem 266:12048-52 |
| Murray, D L; Kohorn, B D (1991) Chloroplasts of Arabidopsis thaliana homozygous for the ch-1 locus lack chlorophyll b, lack stable LHCPII and have stacked thylakoids. Plant Mol Biol 16:71-9 |
| Kohorn, B D; Tobin, E M (1989) A hydrophobic, carboxy-proximal region of a light-harvesting chlorophyll a/b protein is necessary for stable integration into thylakoid membranes. Plant Cell 1:159-66 |
