Transduction of Osmostress Signals by the Yeast Sln1 Protein

对细胞来说，一种普遍的压力是水的可用性的变化，称为渗透压力。古斯汀博士的团队和其他人之前的研究已经在贝克酵母中发现了一条渗透应激信号通路，这条通路由包括Sln1蛋白在内的几个表面渗透传感器控制。尽管人们对各种表面传感器如何检测特定化学物质了解甚多，但对Sln1等蛋白质的渗透传感机制知之甚少。本项目的目的是准确确定细胞表面Sln1蛋白如何将渗透胁迫的外部信号转化为细胞内信号通路的激活。Sln1感知两种类型的渗透胁迫。当细胞外的盐浓度增加（高渗应激）时，Sln1被关闭。当细胞被放入稀释溶液（低渗压力）时，水进入细胞以平衡细胞内较高的盐分。在真菌或植物等具有坚硬外墙的细胞中，这种水的进入会增加内部的压力（称为胀气）。Sln1被肿胀的增加所刺激。为了更好地理解Sln1是如何被刺激的，分离出表现为细胞具有更高膨胀压力的Sln1过度活跃突变体。Sln1通常以一对（二聚体）相同的蛋白质（单体）的形式存在。从这些遗传结果中得出的重要见解是，预计激活Sln1的突变会破坏其单体之间的键，从而允许在Sln1嵌入膜的区域内，一个单体在另一个单体之间滑动。这些数据表明，Sln1信号是由膨胀诱导的蛋白质膜嵌入部分之间的键断裂介导的。为了验证这一假设，该项目将生成并测试额外的突变体，以确定单体之间的键的减弱或增强是否对Sln1活性有预期的影响。此外，本研究将绘制Sln1的膜嵌入区域和膜区域中单体之间的接触点。比较正常和过度活跃的Sln1突变体的这两种特性，将产生Sln1在激活过程中如何改变形状的结构模型。该研究结果将为理解植物和真菌中发现的许多sln1相关传感器蛋白的激活机制提供一个重要的范式。更广泛的影响：来自不同层次的学生将参与这个项目的各个方面。Gustin博士一直在指导本科生和研究生，包括那些来自代表性不足的群体的学生，其中许多人后来从事科学研究、教育和管理工作。Gustin博士每年平均接待10名本科生，他们全年或作为暑期实习生开展子项目。

One universal stress for cells is a change in the availability of water, called osmotic stress. Previous studies by Dr. Gustin's group and others have identified an osmotic stress signaling pathway in Baker's yeast, a pathway that is controlled by several surface osmosensors including the protein Sln1. Although much is known about how various surface sensors detect specific chemicals, very little is known about the mechanism of osmosensing by proteins like Sln1. The purpose of this project is to determine exactly how the cell surface Sln1 protein works to convert the external signal of osmotic stress into activation of signaling pathways inside the cell. Sln1 senses two types of osmotic stress. When the salt concentration outside the cell is increased (hypertonic stress), Sln1 is turned off. When cells are placed into dilute solutions (hypotonic stress), water enters the cell to balance the higher salt inside. In cells with a stiff exterior wall like fungi or plants, this water entry increases the pressure inside (called turgor). Sln1 is stimulated by the increase in turgor. To better understand how Sln1 is stimulated, hyperactive mutants of Sln1 that behaved as if the cell had higher turgor pressure were isolated. Sln1 is normally present as a pair (dimer) of identical proteins (monomers). The important insight from these genetic results is that mutations that activate Sln1 are predicted to break bonds between its monomers, allowing sliding of one monomer by another in the region where Sln1 is embedded in the membrane. These data suggest the hypothesis that Sln1 signaling is mediated by turgor-induced breakage in bonds between the membrane-embedded portions of the protein. To test this hypothesis, this project will generate and test additional mutants to determine whether weakening or strengthening bonds between monomers have the predicted effect on Sln1 activity. In addition, this research will map the membrane-embedded regions of Sln1 and the contact points between the monomers in the region of the membrane. Comparison of these two properties between normal and hyperactive mutants of Sln1 will generate a structural model of how Sln1 changes shape during its activation. Findings from this study will provide an important paradigm for understanding the activation mechanism for the many Sln1-related sensor proteins found in plants and fungi. Broader Impacts: Students from various levels will be involved in various aspects of this project. Dr. Gustin has been mentoring undergraduate and graduates students including those from underrepresented groups, many of whom have gone on to careers in scientific research, education and administration. Every year Dr. Gustin hosts an average of 10 undergraduate students who carry out sub projects either during the whole year or as summer interns.