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被称为细胞壁。细胞壁由互锁的蛋白质和多糖组成，在细胞和环境之间起缓冲作用。在病原真菌中，菌壁对宿主的生存至关重要。因此，参与细胞壁调节和组装的酶是抗真菌药物的极佳靶点。酿酒酵母的孢子壁是在产孢过程中重新形成的复杂结构。孢子壁的组装为研究真菌壁组装过程和协调该过程的信号通路提供了一个很好的系统。Gip1-Glc7磷酸酶复合物介导的信号通路只有在细胞质分裂完成后才启动细胞壁的组装。Gip1蛋白的结构-功能分析将用于探索膜关闭触发Gip1- glc7信号传导的基础。该研究还将阐明Gip1-Glc7调控该通路下游组分转录因子Gis1的机制。虽然真菌细胞壁的成分是已知的，但将这些物质组装成功能性细胞壁的酶却知之甚少。我们已经确定了几个基因编码参与孢子壁构建的候选酶。这些基因突变的表型将被详细描述。此外，这些蛋白质的酶活性将使用体外组装试验进行检查。酿酒葡萄球菌的孢子壁成分也在病原体白色念珠菌的细胞壁中发现，它们对生物体的毒力很重要。我们将研究这些成分是否以类似酿酒葡萄球菌孢子壁的方式组装成白色念珠菌细胞壁。孢子壁保护孢子免受一系列环境损害的显著能力主要是由于孢子壁的外层。这些层有两种主要成分：壳聚糖、氨基葡萄糖多糖和一种含有交联氨基酸二酪氨酸的独特聚合物。这种二酪氨酸聚合物位于孢子壁的最外表面，在一定程度上起着扩散屏障的作用，限制了可溶性分子进出孢子壁的运动。为了更好地了解这种聚合物的功能，二酪氨酸聚合物的结构将使用质谱法和固态13C核磁共振相结合的方法来确定。

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.