Molecular Basis of Hypertonicity Sensing by Yeast Yvc1p

酵母Yvc1p高渗感测的分子基础

基本信息

批准号：
6831700
负责人：
YOSHIRO SAIMI
金额：
$ 28.01万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-05-01 至 2007-11-30
项目状态：
已结题

项目摘要

The basis of how cells detect water stress remains largely obscure and opportunities to study its molecular mechanism are few. Upon hyper-osmotic shock, yeast cells release Ca2+ from their vacuoles to the cytoplasm. We cloned the gene that encodes a cation channel in the vacuolar membrane, Yvclp, that is apparently the conduit for this Ca2+ release. We recently found that physical pressures or osmotic upshocks open this channel under patch clamp, making it possible that the conduit is also the sensor of the force in vivo. This hypothesis will be tested. We will change Yvclp by mutations or use fungal Yvclp homologs. We will then test whether the changes in Yvclp's mechanosensitivity under patch clamp match the changes in Ca2+ release in vivo as measured by aequorin luminometry. Yvclp is activated by Ca2+ itself, and we will use mutants to test whether this activation is important in its function in osmotic defense. Others have observed that vacuoles actively shrink and crenellate upon upshock and found that there is a signal-transduction pathway involving phosphoinositide metabolism. Part of our effort will be to integrate our finding on Yvclp with this shrinkage and crenellation. We will examine whether and how these physical changes relate to the function of Yvclp. We will also improve on the techniques to study Yvclp and osmotic responses, including the possible production of Yvclp in frog oocytes or bacteria, rapid perfusion, and the use "cameleon", as a single-cell Ca2+ reporter.

细胞如何检测水应激的基础在很大程度上晦涩难懂，研究其分子机制的机会很少。高渗透性休克后，酵母细胞从液泡中释放出Ca2+到细胞质。我们克隆了编码液泡膜YVCLP中阳离子通道的基因，该基因显然是该CA2+释放的导管。我们最近发现，物理压力或渗透振荡在斑块夹下打开此通道，使该导管也可能是体内力的传感器。该假设将进行检验。我们将通过突变更改YVCLP或使用真菌YVCLP同源物。然后，我们将测试在贴片夹下YVCLP的机械敏感性的变化是否匹配了通过Aequorin Luminemememememelem seal seal tene ca2+释放的变化。 YVCLP被Ca2+本身激活，我们将使用突变体来测试这种激活在其在渗透防御中的功能中是否重要。其他人则观察到液泡在上垫上积极收缩和旋转酸盐，发现存在涉及磷酸肌醇代谢的信号转导途径。我们的一部分努力是将我们在YVCLP上的发现与这种收缩和潮流相结合。我们将检查这些物理变化是否与YVCLP的功能如何相关。我们还将改进研究YVCLP和渗透反应的技术，包括在青蛙卵母细胞或细菌中可能产生YVCLP，快速灌注和使用“ Cameleon”作为单细胞CA2+报告剂。