Role of a Cellulose Binding Protein in the Dictyostelium Spore Coat

9730036个西方细胞构建细胞外屏障(例如，墙)，将细胞彼此或与外部环境隔开。这种细胞外屏障的一个共同结构主题是，它们由纤维成分和无定形成分组成的网络组成。该项目的目标是更好地了解粘菌盘基菌的孢子皮形成机制，盘基菌是一种真核生物，在动植物分离前后从进化树中分离出来。一个基本的假设是，至少在一般原理方面，关于网柄菌孢子皮是如何由细胞组装的知识将与其他生物的细胞壁生物合成有关，如细菌、植物和其他真菌。网柄担子菌是研究纤维屏障形成的有利系统，原因有很多：在实验室中很容易操作，因为孢子被层在很短的时间内以严格发育调节的方式形成；生物化学上有用的物质很容易分离；生物体在遗传上是易驯化的，大多数外壳蛋白基因已经被克隆；并且，由于产孢子对生活史(在实验室环境中)不是必需的，可以用基因工程设计菌株的外壳基因，这些基因被突变和替换。网柄菌孢子皮含有纤维素纤维、半乳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是被膜蛋白复合体的一部分。SP85含量丰富，存在于被毛组装开始时，与纤维素一起唯一地定位于被毛的内层/中层，由PSPB基因编码，似乎具有模块化结构。在大肠杆菌中表达的SP85和SP85的蛋白结构域表明，纤维素结合是该蛋白C-末端的一种特性。韦斯特博士推测，SP85通过一种交联性机制帮助组织外套中的纤维素和蛋白质成分。对纤维素分解系统中的模块化纤维素结合蛋白以及其他“壁”中的蛋白质的研究表明，生化和遗传学方法将为分析这种超分子复合体提供信息。该项目的第一个目标是通过基因破坏在体内阻断SP85的表达。在一种互补的方法中，N-末端和C-末端结构域将被过度表达，以试图基于假设的交联性产生显性的负面效应。对这些突变体的外壳表型的分析将显示SP85是否如体外结合研究所预期的那样参与组织其他外壳分子。如果有证据表明存在冗余编码功能，将在其他外壳基因缺失突变体的背景下进行干扰。第二个目的是通过生化研究来确定SP85 N-末端和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，并避免将SP85从复合体中分离出来，SP85将通过生长的不表达其他孢子皮蛋白的Dictyostelials细胞来表达。第三个目标是使用表位标签在体内定位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标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.