BIOCHEMICAL ANALYSIS OF THE YEAST A-FACTOR RECEPTOR

酵母 A 因子受体的生化分析

• 批准号: 3294268
• 负责人: GEORGE F. SPRAGUE
• 金额: $ 10.9万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3294268
• 关键词: cell cycle; conformation; dialysis; enzyme linked immunosorbent assay; fungal genetics; fusion gene; gel electrophoresis; gel filtration chromatography; gene expression; genetic manipulation; genetic transcription; high performance liquid chromatography; hormone binding protein; hormone receptor; immunofluorescence technique; intracellular membranes; ion exchange chromatography; laboratory rabbit; mass spectrometry; membrane permeability; membrane proteins; pheromone; phosphorylation; protein sequence; receptor; sedimentation; structural genes; yeasts

Interaction between peptide pheromones and cell surface receptors controls cell division and other physiological events during mating in the yeast Saccharomyces cerevisiae. Haploid cells of the a mating type secrete a peptide pheromone, a factor, to which cells of the yield mating type respond. The response includes changes in transcription of a set of genes and arrest of the cell division cycle in G1. It is believed that a factor binds to a specific receptor on the surface of yield cells and thereby creates an internal signal(s) that triggers the physiological response. Our long-term goal is to understand how binding of pheromone to receptor generates an internal signal and what the specific intracellular targets of that signal are. Thus, we expect to understand the pathway whereby binding of ligand at the cell surface can control complex physiological processes such as cell division. This is a fundamental biological problem. Peptide hormones that control cell division are ubiquitous in eukaryotic organisms. Yeast offer the unique opportunity to combine biochemical and genetic approaches to this problem. We have identified the gene, STE3, that very likely encodes the a-factor receptor. In the proposed work we will determine by direct binding assays whether this gene indeed encodes the receptor. Using a combination of gene fusion methods and antibody probes to different portions of the STE3 product, we will determine the topology of the receptor in the membrane -- what parts of the receptor are extracellular and whether it traverses the membrane seven times as the primary amino acid sequence suggests. We will purify the receptor to determine its subunit composition and to permit detailed analysis of pheromone binding. For instance, using physical techniques, we will determine whether the structure of the receptor changes when pheromone is bound. We will also determine whether the location of the receptor changes when ligand is bound. In particular, do the receptor molecules cluster at one position on the cell surface, and are the receptor-ligand complexes internalized? Finally, we will investigate the nature of the intracellular chemical signal generated by binding of pheromone to receptor.

肽信息素与细胞表面受体的相互作用 酵母交配期间的细胞分裂和其他生理事件 酿酒酵母 a交配型的单倍体细胞分泌a 肽信息素，一种使细胞产生交配型的因子 回答。 这种反应包括一组基因转录的变化 并将细胞分裂周期阻滞在G1期。 据信， 与产量细胞表面上的特异性受体结合， 产生触发生理反应的内部信号。 我们的长期目标是了解信息素与受体的结合 产生一个内部信号， 这些信号是。 因此，我们希望了解结合的途径， 细胞表面的配体可以控制复杂的生理过程 例如细胞分裂。 这是一个基本的生物学问题。 肽 控制细胞分裂的激素在真核生物中普遍存在， 有机体 酵母为联合收割机结合生物化学和 遗传学方法来解决这个问题。 我们已经确定了基因，STE 3，它很可能编码a因子 受体的 在拟议的工作中，我们将通过直接结合测定来确定 这个基因是否真的编码受体。 利用基因组合 STE 3不同部分的融合方法和抗体探针 我们将确定膜上受体的拓扑结构-- 受体的哪些部分是细胞外的，以及它是否穿过 膜的七倍，因为初级氨基酸序列表明。 我们将 纯化受体以确定其亚基组成并允许 信息素结合的详细分析 例如，使用物理 技术，我们将确定受体的结构是否发生变化， 当费洛蒙被束缚的时候 我们还将确定 当配体结合时受体发生变化。 特别是， 分子聚集在细胞表面的一个位置， 受体配体复合物内化？ 最后，我们将调查 细胞内化学信号的性质由结合 信息素到受体