ROLE OF NOVEL KELCH REPEAT PROTEINS IN YEAST DIFFERENTIATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 Heitman实验室研究了一种新的G蛋白受体，该受体允许芽殖酵母细胞感知葡萄糖并进行假菌丝丝状生长。该受体与G蛋白α亚基Gpa 2偶联，Gpa 2具有异源三聚体Ga亚基的所有特征，但不与已知的酵母Gbg亚基Ste 4和Ste 18起作用。我们的研究揭示了两个新的同源蛋白，Gpb 1和Gpb 2，功能作为Gb结构模拟物，以抑制信号传导的Gpa 2和一个未知的目标在信号传导途径。最有趣的是，Gpb 1和Gpb 2蛋白缺乏B亚基特有的7个标记WD-40基序。相反，两者都有7个kelch重复序列，以前涉及蛋白质-蛋白质相互作用。含有7个kelch重复的酶，半乳糖氧化酶的结构是已知的。该蛋白质采用7叶b螺旋桨结构，基本上与Gbg已知的X射线晶体结构重叠。 这些研究揭示了一类新的异源三聚体G蛋白，相关的B亚基模拟物可能在多细胞真核生物中发挥作用。 我们中的一个有FLAG标记的Gpb 1和Gpb 2。Gpb 1-FLAG（C-末端标记的构建体）或FLAG-Gpb 2（N-末端标记的构建体）在gpb 1，2gpa 2三重突变酵母菌株背景中从2 μ m质粒上的减毒ADH 1启动子表达。通过加入FLAG肽洗脱相互作用蛋白。银染和考马斯亮蓝染色均可见特异性条带。目前的工作是FLAG标记的受体和耦合G α亚基Gpa 2。我们预计，这些研究将揭示新的亚基（如γ亚基，我们有一个强有力的候选人），以及相关的调节蛋白和效应。由于对酵母中的Ras-cAMP信号通路有相当多的了解，因此有许多候选者，包括Ras 1, 2，Cap 1，腺苷酸环化酶，Pde 1, 2，Pde 1，2和蛋白激酶A的亚基，这应该可以直接解释质谱相互作用数据。

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. The Heitman lab studies a novel G protein receptor that allows budding yeast cells to sense glucose and undergo pseudohyphal filamentous growth. The receptor is coupled to a G protein alpha subunit, Gpa2, which has all the hallmarks of a heterotrimeric Ga subunit but does not function with the known yeast Gbg subunits Ste4 and Ste18. Our studies revealed two novel homologous proteins, Gpb1 and Gpb2, function as Gb structural mimics to inhibit signaling by Gpa2 and an unknown target in the signaling pathway. Most interestingly, the Gpb1 and Gpb2 proteins lack the 7 signature WD-40 motifs characteristic of b subunits. Instead, both have 7 kelch repeats, previously implicated in protein-protein interactions. The structure of an enzyme containing 7 kelch repeats, galactose oxidase, is known. This protein adopts a 7-bladed b-propeller structure essentially superimposable upon the known X-ray crystal structures of Gbg. These studies reveal a novel class of heterotrimeric G proteins, and related b subunit mimics may function in multicellular eukaryotes. One of us has FLAG-tagged Gpb1 and Gpb2. Gpb1-FLAG (C-terminally tagged construct) or FLAG-Gpb2 (N-terminally tagged construct) were expressed from an attenuated ADH1 promoter on a 2 um plasmid in a gpb1,2 gpa2 triple mutant yeast strain background. Interacting proteins were eluted by adding FLAG peptide. On both silver staining and Coomassie staining, specific bands were apparent. Current work is to FLAG-tag both the receptor and the coupled G alpha subunit Gpa2. We anticipate that these studies will reveal novel subunits (such as the gamma subunit, for which we have a strong candidate) as well as the relevant regulatory proteins and effectors. Since there is quite a bit known about the Ras-cAMP signaling pathway in yeast, there are many candidates, including Ras1/2, Cap1, adenylyl cylase, Ira1/2, Pde1,2, and the subunits of protein kinase A, which should make interpretation of the mass spectrometry interaction data straightforward.