Role of a Cellulose Binding Protein in the Dictyostelium Spore Coat

9730036细胞构建细胞外屏障（如细胞壁）将细胞彼此或与外部环境分离。这种细胞外屏障的一个共同的结构主题是它们由纤维成分和非晶成分组成的网络组成。本项目的目的是为了更好地了解黏菌盘状盘齿龙（Dictyostelium disideum）孢子外壳的形成机制。盘状盘齿龙是一种真核生物，从围绕动植物分离的进化树中分离出来。一个潜在的假设是，至少就一般原理而言，了解盘基骨菌孢子外壳是如何由细胞组装的，将与其他生物（如细菌、植物和其他真菌）的细胞壁生物合成有关。由于多种原因，盘状网柱体是研究纤维素屏障形成的有利系统：由于孢子外壳在短时间内以严格的发育调节方式形成，因此在实验室中很容易操纵外壳的形成；可以很容易地分离出生物化学有用数量的物质；这种生物在遗传上是可控制的，大多数外壳蛋白基因已经被克隆出来；而且，由于孢子在生命周期中不是必需的（在实验室环境中），菌株可以用突变和替换的外壳基因来改造。盘基钢门菌的孢子外壳含有纤维素原纤维、一种谷露聚糖多糖和九种主要蛋白质。韦斯特博士已经证明，其中一种名为SP85的蛋白质能够与纤维素结合。根据早期的研究，SP85是前涂层蛋白复合物的一部分。SP85含量丰富，存在于被毛组装的开始，与纤维素一起独特地定位于被毛的内层/中间层，由pspB基因编码，具有模块化结构。SP85和SP85蛋白结构域在大肠杆菌中的表达表明，纤维素结合是该蛋白c端一半的特性。韦斯特博士推测，SP85通过交联机制帮助组织被毛的纤维素和蛋白质成分。对纤维素分解系统中模块化纤维素结合蛋白的研究，以及其他“壁”中的蛋白质的研究，如单细胞藻类衣藻、枯草芽孢杆菌的孢子外壳和动物基底膜，表明生化和遗传方法将为分析这种超分子复合物提供信息。该项目的第一个目标是通过基因破坏来阻断SP85在体内的表达。在一种互补的方法中，N端和c端结构域将被过度表达，试图基于假设的交联活性产生显性的负面影响。分析这些突变体的被毛表型将显示SP85是否如体外结合研究中预期的那样参与组织其他被毛分子。如果存在冗余编码功能的证据，则将在其他外壳基因缺失突变的背景下进行破坏。第二个目的是通过生化研究确定SP85 N端和C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，并避免将SP85从复合体中分离出来，SP85将通过生长不表达其他孢子外壳蛋白的盘基骨柱细胞来表达。第三个目标将是使用表位标记在体内定位SP85结构域，看看它们是否与第二个目标中确定的假定结合靶点的已知物理分布相关。

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.