9630782 Reddy Calmodulin, a key intracellular calcium receptor, is believed to mediate a number of calcium regulated processes by interacting with and regulating the activity of a number of key enzymes and structural proteins. However, little is known about the proteins that interact with calcium/calmodulin complexes in plants. The lack of information on the calmodulin-binding proteins and their identity has been a major limitation in plants in elucidating calmodulin action at the biochemical and molecular level. This laboratory recently isolated a full-length (4 kb) cDNA encoding a novel calmodulin-binding protein (KCBP, kinesin-like calmodulin-binding protein) from Arabidopsis by screening an expression library with biotinylated calmodulin. The deduced protein is 1261 amino acids long, with a carboxyl-terminal region domain of about 340 amino acids that has significant sequence similarity with the motor domain of kinesin heavy chains and contained putative ATP- and microtubule-binding sites typical of kinesins. By analyzing calmodulin-binding activity of truncated proteins expressed in Escherischia coli, the calmodulin-binding region mapped to a stretch of about fifty amino acid residues near the C-terminus of the protein. This new member of the kinesin superfamily is the first protein shown to be both a calmodulin-binding protein and a kinesin heavy chain. A homologue of KCBP that binds calmodulin has been isolated from potato, suggesting that this protein is ubiquitous in plants. The calmodulin-binding domain in these two proteins is highly conserved and not present in any of more than forty kinesins or kinesin-like proteins thus far characterized from animals. Very little is known about either calmodulin-binding proteins or microtubule motor proteins in plants. The proposed studies are aimed at elucidating the function of this novel protein. KCBP binding to microtubules under various conditions will be analyzed, using either full-length or truncated proteins expressed in and p urified from E. coli. In vitro motility assays will be used to analyze the motor activity of KCBP and selected domains, including the effect of calcium and calmodulin (in collaboration with Dr. Sharyn Endow of Duke University). KCBP will be immunolocalized in various plant tissues and cultured cells at different stages of the cell cycle. The calmodulin-binding domain will be analyzed by fine mapping and determination of the binding affinity. The expression of KCBP will be manipulated, constitutively or in a tissue-specific manner, using sense and antisense constructs. The long term goal is to understand the function of KCBP and the regulatory role of calcium and calmodulin in KCBP-driven motor activity. The proposed studies will enhance our understanding of this unique protein in intracellular transport and help gain new insights into calmodulin function. ***
