Role of a Cellulose Binding Protein in the Dictyostelium Spore Coat

纤维素结合蛋白在盘基网柄菌孢子衣中的作用

• 批准号: 9730036
• 负责人: Christopher West
• 金额: --
• 依托单位: University of Florida
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9730036&HistoricalAwards=false
• 关键词: Role; Cellulose; Binding; Protein; Dictyostelium

9730036 West Cells construct extracellular barriers (e.g., walls) to separate cells from each other or from the external environment. A common structural theme of such extracellular barriers is that they consist of a network of fibrillar components and an amorphous component. The goal of this project is to better understand the mechanism of spore coat formation in the slime mold Dictyostelium discoideum, a eukaryote which diverged from the evolutionary tree around the parting of plants and animals. An underlying assumption is that knowledge of how the Dictyostelium spore coat is assembled by the cell will be relevant, at least in terms of general principles, to cell wall biosynthesis in other organisms, such as bacteria, plants, and other fungi. Dictyostelium is an advantageous system for studying the formation of cellulosic barriers, for a variety of reasons: coat formation is readily manipulable in the laboratory, since the spore coat forms over a short period of time in a strictly developmentally-regulated fashion; biochemically useful quantities of material can be readily isolated; the organism is genetically tractable, and most of the coat protein genes have already been cloned; and, since sporulation is non-essential for the life cycle (in the laboratory setting), strains can be engineered with coat genes can that are mutated and replaced. The Dictyostelium spore coat contains cellulose fibrils, a galuran polysaccharide, and nine major proteins. Dr. West has demonstrated that one of these proteins, SP85, which is known from earlier work to be part of a precoat protein complex, is capable of binding cellulose. SP85 is abundant, is present at the start of coat assembly, is uniquely localized in the inner / middle layers of the coat along with cellulose, is encoded by the pspB gene, and appears to have a modular structure. Expression of SP85 and protein domains of SP85 in E. coli showed that cellulose-binding is a property of the C-terminal half of the protein. Dr. West hypot hesizes that SP85 helps to organize the cellulose and protein components of the coat by a cross-linking mechanism. Studies of modular cellulose-binding proteins in cellulolytic systems, and of proteins in other "walls" such as that of the unicellular alga Chlamydomonas, the spore coat of the bacterium B. subtilis, and animal basement membranes, suggest that biochemical and genetic approaches will be informative for analyzing this supramolecular complex. The first aim of the project is to block expression of SP85 in vivo by gene disruption. In a complementary approach, the N- and C-terminal domains will be overexpressed in an attempt to create a dominant negative effect based on the hypothesized cross-linking activity. Analysis of coat phenotype in these mutants will show whether SP85 is involved in organizing other coat molecules as anticipated from the in vitro binding studies. If there is evidence for redundantly encoded functions, disruptions will be carried out in a background of other coat gene deletion mutants. The second aim is to determine the binding specificities of SP85 N- and C- terminal domains through biochemical studies. To facilitate proper tertiary folding and glycosylation, and to avoid isolating SP85 from a complex, SP85 will be expressed by growing Dictyostelium cells which do not express other spore coat proteins. The third aim will be to use epitope tagging to localize the SP85 domains in vivo, to see if they correlate with the known physical distributions of the putative binding targets identified in the second aim.

9730036西细胞构建细胞外屏障（例如墙壁），以将细胞彼此或外部环境分开。 这种细胞外屏障的一个常见结构主题是，它们由纤维构件网络和无定形成分组成。该项目的目的是更好地了解粘液模具dictyostelium discoideum中孢子涂层形成的机制，这是一种真核生物，与植物和动物分离的进化树不同。 一个基本的假设是，对细胞的组装方式至少在一般原理方面与其他生物体（例如细菌，植物和其他真菌）中的细胞壁生物合成至少相关。由于各种原因，dictyostelium是研究纤维素屏障形成的有利系统：在实验室中很容易操纵外套，因为在短时间内以严格的发育方式形成了孢子涂层；生化有用的材料很容易被隔离；该生物在遗传上是可触犯的，大多数外套蛋白基因已经克隆。而且，由于孢子形成在生命周期（在实验室环境中）不必要，因此可以用突变和替换的外套基因进行菌株设计。 dictyostelium孢子涂层含有纤维素原纤维，galuran多糖和九种主要蛋白质。 West博士证明，这些蛋白质之一Sp85（从早期的工作中是Procoat蛋白质复合物的一部分）都能结合纤维素。 SP85丰富，存在于外套组件的开头，与纤维素一起在外套的内部 /中间层中唯一地定位，由PSPB基因编码，并且似乎具有模块化结构。 大肠杆菌中SP85的SP85和蛋白质结构域的表达表明，纤维素结合是蛋白质的C末端一半的特性。 West Hypot博士heshestes SP85有助于通过交联机制组织外套的纤维素和蛋白质成分。 研究纤维素分解系统中的模块化纤维素结合蛋白，以及其他“壁”中的蛋白质（例如单细胞藻藻衣原体，细菌B.枯草芽孢杆菌的孢子涂层和动物基底膜的孢子）的研究，这表明生物化学方法和遗传学方法将会要分析这种超分子复合物的信息。 该项目的第一个目的是通过基因破坏来阻断体内SP85的表达。 在互补方法中，N-和C末端域将过表达，以试图根据假设的交联活性产生主要的负面影响。 这些突变体中外套表型的分析将表明SP85是否按照体外结合研究预期的是组织其他外套分子。 如果有证据表明编码功能，则将在其他外套缺失突变体的背景下进行中断。 第二个目的是通过生化研究确定SP85 N-和C末端结构域的结合特异性。 为了促进适当的三级折叠和糖基化，并避免从复合物中隔离SP85，SP85将通过不表达其他孢子涂层蛋白的dictyostelium细胞来表达。 第三个目的是使用表位标记在体内定位SP85域，以查看它们是否与第二个目标中确定的假定结合靶标的已知物理分布相关。