Functional Analysis of a Novel Calmodulin-Binding Protein from Arabidopsis

小行星9630782 钙调素是一种重要的细胞内钙受体，它通过与许多关键酶和结构蛋白相互作用并调节其活性来介导许多钙调节过程。 然而，对植物中与钙/钙调素复合物相互作用的蛋白质知之甚少。 缺乏钙调素结合蛋白及其身份的信息一直是在植物中阐明钙调素在生物化学和分子水平上的作用的主要限制。本实验室最近通过筛选生物素化钙调蛋白表达文库，从拟南芥中分离出编码一种新型钙调蛋白结合蛋白（KCBP，kinesin-like calmodulin-binding protein）的全长（4 kb）cDNA。 推导出的蛋白质是1261个氨基酸长，与驱动蛋白重链的马达结构域具有显着的序列相似性，并包含推定的ATP和微管结合位点典型的驱动蛋白的羧基末端区域域的约340个氨基酸。 通过分析截短的蛋白质表达在土方ischia大肠杆菌中的钙调素结合活性，钙调素结合区域映射到一段约50个氨基酸残基附近的蛋白质的C-末端。 这个新的驱动蛋白超家族成员是第一个被证明是钙调蛋白结合蛋白和驱动蛋白重链的蛋白质。 结合钙调素的KCBP同源物已从马铃薯中分离出来，表明这种蛋白质在植物中普遍存在。 这两种蛋白质中的钙调素结合结构域是高度保守的，并且不存在于迄今为止从动物中表征的四十多种驱动蛋白或驱动蛋白样蛋白中的任何一种中。 对植物中的钙调素结合蛋白或微管马达蛋白知之甚少。 这些研究旨在阐明这种新蛋白的功能。 KCBP在不同条件下与微管的结合将被分析，使用在E.杆菌 体外运动试验将用于分析KCBP和选定结构域的运动活性，包括钙和钙调蛋白的作用（与杜克大学的Sharyn Endow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。 将通过精细作图和测定结合亲和力来分析钙调素结合结构域。 将使用正义和反义构建体，组成型地或以组织特异性方式操纵KCBP的表达。 长期目标是了解KCBP的功能以及钙和钙调素在KCBP驱动的运动活动中的调节作用。 拟议的研究将增强我们对这种独特的蛋白质在细胞内转运的理解，并有助于获得钙调素功能的新见解。 ***

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. ***