ADENYLATE CYCLASE-ASSOCIATE COMPLEXES IN SCHIZOSACCHAROMYCES POMBE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们的实验室已经在分裂酵母中发现了激活腺苷环化酶所需的几个基因的突变。这些基因编码异源三聚体G蛋白的亚基和可能的GPCR，以及三个以前未知的与cAMP信号相关的基因。我们还收集了一系列腺苷环化酶基因的突变，这些突变似乎影响激活，但不影响催化活性。我们想通过研究分裂酵母中与腺苷环化酶物理相关的蛋白质作为碳源环境的函数来补充我们的遗传分析。我们还想研究腺苷环化酶内突变对这些相互作用的影响。我们已经在其内源位点构建了一个完整的带有TAP标签的腺苷环化酶形式，并能够通过Western分析检测到它。我们现在正在使用酵母资源中心的“TAP标记蛋白的纯化”方案，以便从该菌株中分离出蛋白质复合体。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our lab has identified mutations in several genes required for adenylate cyclase activation in fission yeast. These genes encode the subunits of a heterotrimeric G protein and a likely GPCR, along with three genes not previously known to be associated with cAMP signaling. We also have a collection of mutations in the adenylate cyclase gene that appear to affect activation but not catalytic activity. We would like to complement our genetic analyses with a study of the proteins physically associated with adenylate cyclase in fission yeast as a function of the carbon source environment. We would also like to study the effect of mutations within adenylate cyclase on these interactions. We have constructed an integrated TAP-tagged form of adenylate cyclase at its endogenous locus and are able to detect it by Western analysis. We are now using the "Purification of TAP-Tagged Proteins" protocol from the Yeast Resource Center to allow us to isolate protein complexes from this strain.