Structure-function Analysis of the SP85/PsB Spore Coat Protein in Dictyostelium

细胞壁对许多微生物、植物和动物的生命是必不可少的。包括纤维素在内的多糖是许多壁材的主要成分。对动物、衣藻和Volvox富含蛋白质的壁的研究表明，细胞-基质和基质-基质的相互作用是由涉及离散蛋白结构域的高度特异的接触所介导的。这个项目的目标是研究网柄柄菌的孢子衣，以便从长远来看，了解蛋白质在富含纤维素壁的组装中所起的关键作用。与许多细胞壁一样，孢子被层由中心的富含纤维素区组成，两侧被富含蛋白质的层包围。主要的外壳蛋白之一，SP85或PSB，是协调外壳中纤维素和蛋白质相互作用的主要因素。SP85位于细胞膜的内层，在体外可以同时与纤维素和另一种外壳蛋白SP65结合。根据SP85基因敲除菌株的表型和单个结构域过表达的菌株，SP85被认为对纤维素合成的时间(早期功能)和外层的组织(包括蛋白质的结合和纤维素的限制(晚期功能))都有贡献。一种结构是N端和富含半胱氨酸的C1域的融合，它在85%的细胞中阻止纤维素合成所需的检查点，并在15%的细胞中破坏外层。为了确定SP85与纤维素和SP65的结合在体内的作用，计划进行以下实验：1)在生长细胞中表达的全长SP85的基因，从其他外壳蛋白中分泌出来，将在其C1域受到随机突变，以特异性地阻断与纤维素或SP65的结合；2)将定位并用于监测正常和突变的SP85的结构；将目标1中定义的失活突变引入到在前孢子细胞中表达的SP85和NC1中，并整合到外壳中。表型分析将评估早期和晚期依赖SP85的功能是如何受到影响的。这些结果有望提供有关SP65如何调节SP85在检查点抑制中的作用，SP65在连接内层和外层蛋白质中的假想适配器作用，以及纤维素结合是否对SP85定位和/或在系留和组织纤维素微纤维中重要的信息。如果时间允许，还将进行另一组实验，旨在测试阻断SP65结合的突变的特异性。这些方法将测试体外研究定义的特定结构域活动的体内相关性，从而避免单独使用部分长度表达构建体所固有的潜在伪像。纤维素-SP85-SP65三聚体被预测为一个外壳组装核心模块，可以在未来的研究中扩展，以了解SP85和其他外壳蛋白中其他离散的蛋白质结构域是如何组织壁组装的。广泛的影响：将为SP85富含半胱氨酸的结构域定义的结构-功能关系有望推广到在植物和具有经济和健康意义的真核微生物中发现的其他富含纤维素壁的相关结构域。最终，这些研究可能会为纤维素合成酶是如何调控的，纤维素是如何结晶的，纤维素纤维是如何限制在外层蛋白质层内的，以及细胞壁层是如何形成的，建立了有用的先例。该项目将继续吸收研究生、本科生和高中生，包括妇女和任职人数不足的少数群体，作为研究受训人员参与，以实现与国际和平协会在其学术环境中的教育承诺相一致的目标。将与美国和国际上的核心实验室和其他研究实验室进行重大互动。这些活动将有助于蛋白质组学、糖生物学和细胞壁生物学方面的总体努力，并有望成为整个大学社区的持续资源。所有研究结果将通过会议和出版物及时与科学界分享。

Cell walls are essential for the life of many microbial eukaryotes, plants and animals. Polysaccharides including cellulose are a major component of many walls. Studies of protein-rich walls in animals, Chlamydomonas and Volvox have shown that cell-matrix and matrix-matrix interactions are mediated by highly specific contacts involving discrete protein domains. The goal of this project is to study the spore coat of Dictyostelium in order to understand, in the long term, the critical roles played by proteins in the assembly of cellulose-rich walls. Like many cell walls, the spore coat consists of a central cellulose-rich region surrounded on either side by protein-rich layers. One of the major coat proteins, SP85 or PsB, is a prime suspect for coordinating cellulose- protein interactions in the coat. SP85 is located in the inner layer of the coat near the plasma membrane, and can simultaneously bind cellulose and another coat protein, SP65, in vitro . Based on the phenotypes of SP85 knockout strains and strains in which individual domains have been overexpressed, SP85 is thought to contribute to both the timing of cellulose synthesis (early function) and the organization of the outer layer including incorporation of proteins and confinement of cellulose (late function). One construct, a fusion of the N-terminal and cysteine-rich C1-domains, blocks a checkpoint required for cellulose synthesis in 85% of the cells, and disrupts the outer layer in the 15% that break through the block. The C1-domain uniquely binds both cellulose and SP65.To determine the role of binding of SP85 to cellulose and SP65 for function in vivo, the following experiments are planned: 1), the gene for full-length SP85 expressed in growing cells, from which it is secreted away from other coat proteins, will be subjected to random mutagenesis in its C1-domain to specifically block either cellulose-or SP65-binding; 2), the disulfide-bonding and glycosylation patterns of the C1-domain and nearby mucin-like domains will be mapped and used to monitor the structures of normal and mutant SP85s; and 3), the inactivating mutations defined in aim 1 will be introduced into SP85 and NC1 expressed in prespore cells and incorporated into the coat. Phenotype analyses will assess how the early and later SP85-dependent functions are affected. The results are expected to provide information about how SP65 regulates SP85 action in checkpoint inhibition, the hypothesized adaptor role for SP65 in bridging inner-and outer-layer proteins, and whether cellulose-binding is important for localizing SP85 and/or in tethering and organizing cellulose microfibrils. An additional set of experiments, aimed at testing the specificity of mutations that block SP65-binding, will also be carried out if time permits. These approaches will test the in vivo relevance of specific domain activities defined by in vitro studies, therefore avoiding potential artifacts intrinsic to the sole use of partial-length expression constructs. The cellulose-SP85-SP65 trimer is predicted to be a coat assembly core module which can be extended in future studies to understand how discrete protein domains elsewhere in SP85 and in other coat proteins function to organize wall assembly.Broader impacts: The structure-function relationships to be defined for the cysteine-rich domains of SP85 are expected to be generalizable to related domains of other cellulose-rich walls found in plants and eukaryotic microbes of economic and health significance. Ultimately, these studies are likely to establish useful precedents for how cellulose synthase is regulated, how cellulose is crystallized, how cellulose fibrils are constrained within the outer protein layer, and how cell wall layers are formed. The project will continue to engage the participation of graduate,undergraduate and high school students, including women and underrepresented minorities,as research trainees in execution of the aims consistent with the educational commitment of the PI in his academic setting. There will be significant interaction with core laboratories and other research laboratories in both the USA and internationally. These activities will contribute to the overall critical mass of effort in proteomics, glycobiology and cell wall biology, and is expected to constitute a continuing resource for the overall university community. All findings will be shared with the scientific community in timely fashion via meetings and publications.