Transduction of Osmostress Signals by the Yeast Sln1 Protein

对细胞来说，一种普遍的压力是水分供应的变化，称为渗透胁迫。古斯汀博士的团队和其他人之前的研究已经在贝克氏酵母中发现了渗透胁迫信号通路，该通路由包括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知之甚少，但对SLn1等蛋白质的渗透感知机制知之甚少。这个项目的目的是确切地确定细胞表面SLN1蛋白是如何将渗透胁迫的外部信号转换为激活细胞内的信号通路的。SLN1感受两种类型的渗透胁迫。当细胞外的盐浓度增加(高渗胁迫)时，SLn1被关闭。当细胞被置于稀溶液(低渗压力)中时，水进入细胞以平衡细胞内较高的盐分。在真菌或植物等外壁僵硬的细胞中，这种水分进入会增加细胞内的压力(称为膨胀物)。Sln1受膨胀率增加的刺激。为了更好地了解SLn1是如何被刺激的，我们分离了SLn1的高度活跃的突变体，这些突变体的行为就像细胞具有更高的膨胀压力。SLN1通常以一对(二聚体)相同的蛋白质(单体)存在。从这些遗传结果中得到的重要启示是，激活Sln1的突变预计会打破其单体之间的键，允许一个单体在SLn1嵌入膜的区域与另一个单体滑动。这些数据表明，SLn1信号是由膨压引起的蛋白质膜嵌入部分之间的键断裂所介导的。为了验证这一假设，该项目将产生并测试更多的突变体，以确定减弱或加强单体之间的键是否会对SLn1活性产生预期的影响。此外，本研究还将绘制SLn1的膜包埋区和膜区域中单体之间的接触点。对SLn1正常突变体和超活性突变体之间这两种性质的比较将产生一个结构模型，说明SLn1在激活过程中如何改变形状。这项研究的发现将为理解植物和真菌中发现的许多与SLn1相关的传感器蛋白的激活机制提供重要的范例。更广泛的影响：来自不同层次的学生将参与到这个项目的各个方面。古斯汀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，他们中的许多人后来进入了科学研究、教育和管理领域。每年，古斯汀博士平均招收10名本科生，他们要么在全年开展子项目，要么以暑期实习生的身份参加。

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.