G PROTEINS AND SIGNAL TRANSDUCTION IN NEUROSPORA CRASSA

异源三聚体（α-β-γ）G蛋白是许多人所必需的。 真核生物对环境刺激的反应在丝状 在真菌粗糙脉孢菌中，存在三种已知的G α蛋白（GNA-1和GNA-2）。 GNA-3）和一个G β亚基（GNB-1）。GNA-1和GNA-2有重叠 具有调节雌性生育力和营养生长的功能。只有GNA-1 调节GTP依赖性腺苷酸环化酶活性（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AC的正调节器。这些和其他调查结果 表明cAMP对营养功能很重要，但对性功能不重要。 GNA-1是第一个可以被分类为微生物的Galpha。 哺乳动物家族（Ga/i）。Deltagna-1的系统测试 进行几种哺乳动物Ga基因的互补。的 结果还支持了性和植物性的不同信号通路 生长在N。粗鲁。Deltagnb-1菌株大大减少了GNA-1，但 GNA-2水平正常在其他系统中已经表明， 调节转录。β-γ蛋白调节磷脂酶C (PLC)在哺乳动物中，导致细胞内储存的Ca/2+释放。 液泡是N. crassa;和deltagnb-1 菌株具有空泡缺陷。因此，GNB-1可以调节gna-1， 转录和/或Ca/2+动员。粗鲁。所以我们 假设G蛋白差异调节1）cAMP水平，2） 液泡功能和3）营养期和有性期基因表达 粗糙脉孢菌Neurospora crassa我们的目标是：1）直接 GNA-1和AC之间的相互作用将使用重组进行测试 免疫共沉淀、亲和层析和双杂交 测定。GNA-1和GNA-2的未知目标将由两个- 杂交筛选和遗传抑制分析。GNA-1和GNA-2的作用 在信息素的反应将被测试。2)AC、PDE和cAMP水平 将在哺乳动物Ga转化体中测量。相互作用 哺乳动物Galalpha和GNB-1之间的关系将在双杂交中进行测试。 测定。互补基因和非互补基因之间的嵌合体将是 构造来定义重要的植物或性的区域 功能协调发展的3)将测量AC和PDE活性，以探测 GNA-3在cAMP代谢中的作用GNA-3与其他蛋白质的相互作用 将使用重构、亲和层析和双- 混合分析4)转录与翻译后调节 GNA-1的水平将被GNB-1测试。AC、PDE和cAMP水平将在 在Δ Nb-1菌株中测量。将通过以下方式评估肺泡功能： 测定精氨酸和Ca/2+池及液泡ATP酶活性 将在Δ Nb-1突变体中测试活性和Ca/2+代谢。 GNB-1和其他蛋白质之间的相互作用将使用两个定义- 杂交测定。这些研究将阐明G蛋白信号通路 在丝状真菌中，加速哺乳动物Galpha的功能分析 并深入了解G蛋白的进化。此外，由于同系物 瑟菌的crassa G α基因调节几种丝状菌的毒力 物种，这些调查也将阐明G蛋白的作用， 在真菌发病机制，导致新的疗法，为新兴的真菌 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.