G PROTEINS AND SIGNAL TRANSDUCTION IN NEUROSPORA CRASSA

异三聚体(α-β-伽马)G蛋白对许多人来说是必不可少的 真核生物对环境刺激的反应。在丝状物中 真菌粗糙脉孢菌有三种已知的Galpha蛋白(GNA-1和GNA-1) GNA-3)和一个Gbeta亚基(GNB-1)。GNA-1和GNA-2有重叠 对女性生育和营养生长的调节作用。仅GNA-1 调节GTP依赖的腺酰环化酶活性(AC)，而GNA-1和 GNA-2均影响cAMP磷酸二酯酶活性(PDE)。GNA-3调节 营养生长，但不是性周期所必需的。GNA-3是 被认为是交流的正向调节器。这些和其他发现 表明cAMP对植物性功能很重要，但对性功能不重要。 GNA-1是第一个可以被归类为 哺乳动物家族(Galpha/i)。Delagna-1的系统测试 几个哺乳动物的Galpha基因进行了互补。这个 研究结果也支持有性和植物性的不同信号通路 在粗毛拟青霉中的生长。Delagnb-1菌株极大地降低了GNA-1，但 GNA-2水平正常。在其他系统中已经表明，贝塔-伽马 调节转录。β-γ蛋白调节磷脂酶C (PLC)，导致细胞内存储的钙/2+释放。 N.crassa和deltag nb-1的主要Ca/2+储存库是液泡 菌株有液泡缺陷。因此，GNB-1可能调节GNA-1 转录和/或Ca/2+动员，在粗毛拟青霉。因此，我们 假设G蛋白对cAMP水平有不同的调节作用，2) 液泡功能和3)营养和有性生殖过程中的基因表达 粗枝脉孢菌的发育我们的目标是：1)直接 GNA-1和AC之间的相互作用将通过重建进行测试 检测、免疫共沉淀、亲和层析和双杂交 化验。GNA-1和GNA-2的未知目标将通过两个- 杂交筛选和基因抑制分析。GNA-1和GNA-2的作用 在信息素反应过程中，将对其进行测试。2)AC、PDE和cAMP水平 将在哺乳动物的Galpha转化体中进行测量。互动 哺乳动物Galpha‘s和GNB-1之间将进行双杂交测试 化验。互补和非互补基因之间的嵌合体将是 构建为定义对植物性或有性的重要区域 功能。3)AC和PDE活性将被测量以探索其作用 GNA-3参与cAMP代谢。GNA-3与其他蛋白质的相互作用 将通过重组、亲和层析和两种- 混合分析。4)转录调控与翻译后调控 将测试GNB-1对GNA-1水平的影响。AC、PDE和cAMP水平将是 用delagnb-1菌株测量。将通过以下方法评估液泡功能 精氨酸和Ca/2+池及液泡膜ATPase活性的测定 将在deltag nb-1突变体中测试活性和钙/2+代谢。 GNB-1与其他蛋白质之间的相互作用将使用两个- 混合化验。这些研究将阐明G蛋白信号通路 在丝状真菌中，加快哺乳动物Galpha的功能分析 并洞察G蛋白的进化。此外，由于同源基因 粗毛奈瑟氏菌Gα基因对几种丝状菌的毒力调节 物种，这些研究也将阐明G蛋白的作用 在真菌发病机制方面，导致了新出现的真菌的治疗方法 病原体。

Heterotrimeric (alpha-beta-gamma) G proteins are essential for many responses to environmental stimuli in eukaryotes. In the filamentous fungus Neurospora crassa, there are three known Galpha proteins (GNA-1 and GNA-3) and one Gbeta subunit (GNB-1). GNA-1 and GNA-2 have overlapping functions in regulating female fertility and vegetative growth. Only GNA-1 modulates GTP-dependent adenylyl cyclase activity (AC), while GNA-1 and GNA-2 both affect cAMP phosphodiesterase activity (PDE). GNA-3 regulates vegetative growth, but is not necessary for the sexual cycle. GNA-3 is implicated as a positive regulator of AC. These and other findings indicated that cAMP is important for vegetative, but not sexual functions. GNA-1 was the first microbial Galpha that could be classified in a mammalian family (Galpha/i). Systematic testing of deltagna-1 complementation by several mammalian Galpha genes was performed. The results also support separate signaling pathways for sexual and vegetative growth in N. crassa. Deltagnb-1 strains have greatly reduced GNA-1, but normal GNA-2 levels. It has been shown in other systems that beta-gamma regulates transcription. Beta-gamma proteins regulate phospholipase C (PLC) in mammals, leading to release of Ca/2+ from intracellular stores. The vacuole is the major Ca/2+ reservoir in N. crassa; and deltagnb-1 strains have vacuolar defects. Hence, GNB-1 may regulate gna-1 transcription and/or Ca/2+ mobilization, in N. crassa. Therefore, we hypothesize that G proteins differentially regulate 1) cAMP levels, 2) vacuolar function and 3) gene expression during vegetative and sexual development in Neurospora crassa. Our objectives are: 1) A direct interaction between GNA-1 and AC will be tested using reconstitution assays, co-immunoprecipitation, affinity chromatography and two-hybrid assays. Unknown targets of GNA-1 and GNA-2 will be identified by two- hybrid screens and genetic suppression analysis. Roles of GNA-1 and GNA-2 during the pheromone responses will be tested. 2) AC, PDE and cAMP levels will be measured in the mammalian Galpha transformants. Interactions between the mammalian Galpha's and GNB-1 will be tested in two-hybrid assays. Chimeras between complementing and non-complementing genes will be constructed to define regions important for vegetative or sexual functions. 3) AC and PDE activity will be measured to probe a role for GNA-3 in cAMP metabolism. Interactions between GNA-3 and other proteins will be determined using reconstitution, affinity chromatography and two- hybrid analysis. 4) Transcriptional versus post-translational regulation of GNA-1 levels by GNB-1 will be tested. AC, PDE and cAMP levels will be measured in deltagnb-1 strains. Vacuolar function will be assessed by measuring arginine and Ca/2+ pools and vacuolar ATPase activity PLC activity and Ca/2+ metabolism will be tested in deltagnb-1 mutants. Interactions between GNB-1 and other proteins will be defined using two- hybrid assays. These studies will elucidate G protein signaling pathways in filamentous fungi, speed the functional analysis of mammalian Galpha's and yield insight into G protein evolution. Furthermore, since homologues of N. crassa G alpha genes modulate virulence in several filamentous species, these investigations will also illuminate the role of G proteins in fungal pathogenesis, leading to new therapies for emerging fungal pathogens.