A number of molecules have recently been described that effect the correct transport and assembly of cytoplasmically synthesized proteins to cellular membranes. Few studies, however, have described factors involved in the post-translational insertion of proteins into the chloroplast thylakoid membrane. To identify proteins that bind or modify other proteins during the process of membrane translocation, we developed a new yeast selection scheme that employs the yeast transcriptional activator, GAL4. This selection facilitates the isolation of cDNAs that encode binding proteins, proteases, or post-translational modifiers specific for known target peptide sequences. With this selection scheme we isolated an Arabidopsis cDNA encoding a 596 amino acid polypeptide (termed PRO25) that can genetically interact with the amino terminus of a light harvesting chlorophyll binding protein (LHCP), a cytoplasmically synthesized protein that is post-translationally inserted into the thylakoid membrane. The Arabidopsis CDNA encodes a protein with at least two functional domains, one with similarity to the family of serine/threonine kinases and another that contains an epidermal growth factor (EGF) repeat, the latter of which has not been previously reported in the plant kingdom. Nucleic acid hybridization and antibody staining indicate that this gene and its product are present in other genera of higher plants. The mRNA is expressed in green leaves but not in other plant tissues or in etiolated plants. Preliminary results with polyclonal antisera indicate that the encoded protein may be present on both the plasma membrane and thylakoid membrane of leaf cells, suggesting an intriguing interaction between the two compartments. This proposal addresses the following questions: 1, where in the cell is PRO25 localized?; 2, is the kinase or amino-terminal domain important for interaction with LHCP?; and 3, what is the function of PRO25? The first question will be addressed using immunoelectron microscopy and Western blotting. These studies will include developmental profiles of expression, and the use of control antisera to known proteins of defined location. It must also be determined whether the antisera are specific to one protein or to a family whose members are in multiple locations. The second will be addressed using the yeast functional assay and expression in E. coli to dissect the two protein domains. The third will be addressed by expression and analysis of dominant mutations of PRO25 in transformed Arabidopsis, which should provide a phenotype that will provide a clue to its biological role. %%% This project is the result of a totally unexpected finding. As such, it is highly speculative, and although it is likely that the research will lead to something very interesting and important, it is not yet possible to predict what that "something" will be. The investigator was studying the problem of how cytoplasmically- encoded plant proteins get imported and assembled into chloroplasts, and developed a clever genetic scheme which was intended to fish out genes and gene products which interacted with a specific imported chloroplast protein, the Light Harvesting Chlorophyll a/b Binding Protein. It was expected that this scheme would identify LHCP-specific proteases responsible for LHCP maturation in the chloroplast. Instead, the gene that was fished out by the selection scheme had no resemblance to a protease, but rather had two domains, one of which appears to be a serine/threonine kinase (i.e., an enzyme that adds phosphate residues to serine and threonine residues of proteins) and the other of which appears to be homologous to the epidermal growth factor of animals (which had never been described in plants before). Expression of this protein appears to be light-dependent, suggesting a functional link to photosynthesis. Homologous proteins have been found in other higher plants, suggesting a highly conserved function. Protein phosphorylation is a major mechanism for cellular regulation of protein functions, and the LHCP is known to get phosphorylated in the chloroplast. The question is, what is the function of this curious, novel, plant protein? Depending on the answers to the questions addressed in this research project, there is the potential to learn some very unexpected and important things about how higher plants convert sunlight into foodstuff.
