Functional Analysis of a Novel Calmodulin-Binding Protein from Arabidopsis

9630782红色钙调蛋白是细胞内一种关键的钙受体，通过与一些关键酶和结构蛋白相互作用并调节其活性，被认为参与了一系列钙调节过程。然而，人们对植物中与钙/钙调蛋白复合体相互作用的蛋白质知之甚少。缺乏钙调蛋白结合蛋白及其特性的信息一直是植物在生物化学和分子水平上阐明钙调蛋白作用的主要限制因素。本实验室最近通过筛选含有生物素化钙调蛋白的表达文库，从拟南芥中分离到一个编码新的钙调蛋白结合蛋白(KCBP，kinesin-like calmodin-bindingProtein)的全长(4kb)的基因。该蛋白全长1261个氨基酸，其羧基末端区域约340个氨基酸，与运动蛋白重链的运动区序列具有显著的相似性，并含有典型的运动蛋白的ATP和微管结合位点。通过分析在大肠杆菌中表达的截短蛋白的钙调素结合活性，钙调素结合区位于蛋白C末端附近约50个氨基酸残基的长度上。这个运动蛋白超家族的新成员是第一个被证明既是钙调蛋白结合蛋白又是运动蛋白重链的蛋白质。从马铃薯中分离到一种与钙调蛋白结合的KCBP同源蛋白，这表明该蛋白在植物中普遍存在。这两种蛋白中的钙调蛋白结合域是高度保守的，不存在于迄今为止动物所特有的40多种动蛋白或类似动蛋白中的任何一种。人们对植物中的钙调蛋白结合蛋白或微管马达蛋白知之甚少。拟议的研究旨在阐明该新蛋白的功能。使用在大肠杆菌中表达和纯化的全长或截短的蛋白，将分析在不同条件下KCBP与微管的结合。体外动力分析将用于分析KCBP和选定结构域的运动活性，包括钙和钙调蛋白的影响(与杜克大学的Sharyn Enow博士合作)。KCBP将在细胞周期的不同阶段免疫定位于各种植物组织和培养细胞中。钙调素结合域将通过精细作图和结合亲和力的测定来分析。KCBP的表达将利用正义和反义构建物，以结构性或组织特异性的方式进行操纵。长期目标是了解KCBP的功能以及钙和钙调蛋白在KCBP驱动的运动活动中的调节作用。建议的研究将加强我们对这种独特的蛋白质在细胞内运输的理解，并有助于对钙调蛋白功能的新见解。***

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. ***