Assembly and Function of the Yeast Spore Wall

描述（由申请人提供）：真菌细胞的细胞外基质称为细胞壁。细胞壁由互锁的蛋白质和多糖组成，并作为细胞与其环境之间的缓冲区。在病原真菌中，壁对于在宿主中的生存至关重要。因此，参与细胞壁调节和组装的酶是抗真菌药物的极好靶点。酿酒酵母的孢子壁是在孢子形成过程中重新形成的复杂结构。孢子壁的组装提供了一个很好的系统来研究真菌壁组装的过程和协调该过程的信号通路。Gip 1-Glc 7磷酸酶复合物介导的信号转导途径，启动组装后的细胞壁完成胞质分裂。Gip 1蛋白的结构-功能分析将用于探索膜闭合触发Gip 1-Glc 7信号传导的基础。Gip 1-Glc 7调节途径中的下游组分，转录因子Gis 1的机制也将被阐明。 虽然真菌细胞壁的成分是已知的，但将这些材料组装成功能性壁的酶却知之甚少。我们已经确定了几个基因编码的候选酶参与建设的孢子壁。这些基因中突变体的表型将被详细描述。此外，将使用体外组装测定法检查这些蛋白质的酶活性。对S.在病原体C的细胞壁中也发现了酿酒酵母。白色念珠菌，其中它们对生物体的毒力很重要。我们将检查这些组件是否组装到C中。albicans细胞壁与S.酿酒酵母孢子壁。 孢子壁保护孢子免受一系列环境侵害的显着能力主要是由于孢子壁的外层。这些层有两个主要成分，壳聚糖，葡萄糖胺多糖，和一种独特的聚合物含有交联的氨基酸二酪氨酸。这种双酪氨酸聚合物存在于孢子壁的最外表面上，并部分用作限制可溶性分子进出壁的扩散屏障。为了更好地理解该聚合物如何起作用，将使用质谱法和固态13 C NMR的组合来确定双酪氨酸聚合物的结构。 公共卫生相关性：真菌细胞壁作为真菌细胞与其环境之间的屏障。壁对于真菌病原体逃避宿主免疫应答是必不可少的，并且抗真菌药物通常通过干扰壁合成来起作用。本研究的重点是面包酵母，酿酒酵母，作为真菌壁组装模型的孢子壁的组装。实验的目的是了解调节，组装途径和壁组件的结构。

DESCRIPTION (provided by applicant): The extracellular matrix of fungal cells is referred to as the cell wall. The cell wall is composed of interlocked proteins and polysaccharides and serves as a buffer between the cell and its environment. In pathogenic fungi, the wall is critical for survival in the host. Enzymes involved in the regulation and assembly of the cell wall are therefore excellent targets for antifungal drugs. The spore wall of Saccharomyces cerevisiae is a complex structure that forms de novo during the process of sporulation. Assembly of the spore wall provides an excellent system to investigate both the process of fungal wall assembly and the signaling pathways that coordinate that process. The Gip1-Glc7 phosphatase complex mediates a signaling pathway that initiates assembly of the wall only after the completion of cytokinesis. A structure-function analysis of the Gip1 protein will be used to explore the basis by which membrane closure triggers Gip1-Glc7 signaling. The mechanism by which Gip1-Glc7 regulate a downstream component in the pathway, the transcription factor Gis1, will also be elucidated. Though the constituents of the fungal cell wall are known, the enzymes that assemble these materials into a functional wall are little understood. We have identified several genes encoding candidate enzymes involved in construction of the spore wall. These phenotypes of mutants in these genes will be characterized in detail. Additionally, the enzymatic activities of these proteins will be examined using an in vitro assembly assay. Components of the spore wall in S. cerevisiae are also found in the cell wall of the pathogen C. albicans where they are important for virulence of the organism. We will examine if these components are assembled into the C. albicans cell wall a similar fashion as in the S. cerevisiae spore wall. The remarkable ability of the spore wall to protect the spore from an array of environmental insults is due primarily to the outer layers of spore wall. These layers have two major constituents, chitosan, a glucosamine polysaccharide, and a unique polymer containing the crosslinked amino acid dityrosine. This dityrosine polymer is found on the outermost surface of the spore wall and acts in part as a diffusion barrier that limits the movement of soluble molecules into and out of the wall. To better understand how this polymer functions the structure of the dityrosine polymer will be determined using a combination of mass spectrometry and solid state 13C NMR. PUBLIC HEALTH RELEVANCE: Fungal cell walls act as a barrier between the fungal cell and its environment. The wall is essential for a fungal pathogen to evade the host immune response and antifungal drugs commonly act by interfering with wall synthesis. This study focuses on the assembly of the spore wall of baker's yeast, Saccharomyces cerevisiae, as a model for fungal wall assembly. Experiments are targeted towards understanding the regulation, assembly pathways, and structure of wall components.