Heterotrimeric G Protein-Mediated Cellular Polarization in Yeast

细胞极化对形态发生、免疫反应、神经元发育、趋化性和运动性至关重要。芽殖酵母（Saccharomyces cerevisiae）是一种被广泛研究的真核生物模型，它表现出许多极化现象，包括信号诱导的细胞形状和核位置的变化。在酿酒酵母的生命周期中，两个交配类型相反的单倍体细胞MATa和MATa可以结合形成MATa/a二倍体细胞。每种交配类型组成分泌一种肽交配信息素，将相反类型的营养性生长细胞转化为配子。为了准备单倍体进行细胞和核融合，信息素触发了交配特定基因的诱导，在细胞周期的G1期停止，向交配伴侣极化生长，核迁移到交配投射的尖端。交配信号传递的分子机制已经被很好地理解。信号从质膜到细胞质的传递是由受体偶联异三聚体G蛋白介导的。当被配体占据时，信息素受体通过鸟嘌呤核苷酸交换和伴随的Ga？GTP来自Gbg二聚体（由STE4和STE18编码）。然后信号通过Gbg传输到有丝分裂原？活化蛋白激酶级联。MAP激酶模块由Ste11 （MEKK）、Ste7 （MEK）和Fus3 （MAPK）组成。除了刺激交配信号外，Gbg还起到位置提示的作用。它将双重功能蛋白Far1引导到生长部位。Far1起支架的作用。它汇集了刺激肌动蛋白细胞骨架极化的元素。在生理条件下，Gbg-Far1复合体被认为聚集在细胞表面信息素浓度最高的区域，并标记该区域生长。因此，细胞向信息素的来源方向生长。这被称为趋化性。最近的研究结果表明，信息素响应的Ga蛋白Gpa1在信号诱导极化和核运动的控制中起着重要作用。在对信息素有反应的细胞中，Gpa1直接与交配特异性MAP激酶Fus3和Kar3相互作用，Kar3是一种对交配过程中的核运动至关重要的激酶样蛋白。Gpa1-Fus3相互作用的破坏导致趋化性和核迁移的缺陷。本研究的目的是确定哪些趋化因子依赖于Gpa1对Fus3的募集，并确定Gpa1如何影响交配过程中的核运动。在NSF资助的工作中，将验证以下假设：Gpa1共同定位Fus3和Kar3，使Fus3激活Kar3。由于目前尚无Ga蛋白或MAP激酶调控运动蛋白的先例，也不清楚运动蛋白是如何被激活的，因此这项工作对微管相关运动蛋白的研究具有重要的价值。这项研究的更广泛的影响是，它将增强我们对趋化性和核运动的理解，这是细胞功能的两个基本方面。所有层次的学生都将参与这项研究。

Cellular polarization is essential to morphogenesis, immune response, neuronal development, chemotropism, and motility. The budding yeast, Saccharomyces cerevisiae, is a well studied model eukaryote that exhibits numerous polarization phenomena, including signal-induced changes in cell shape and nuclear position. In the life cycle of S. cerevisiae, two haploid cells of opposite mating type, MATa and MATa, can conjugate to form a MATa/a diploid cell. Each mating type constitutively secretes a peptide mating pheromone that transforms vegetatively growing cells of opposite type into gametes. To prepare haploids for cellular and nuclear fusion, pheromone triggers the induction of mating specific genes, arrest in the G1 phase of the cell cycle, polarized growth toward the mating partner, and nuclear migration to the tip of the mating projection. The molecular mechanisms underlying transmission of the mating signal are well understood. Communication of the signal from the plasma membrane to the cytoplasm is mediated by a receptor-coupled heterotrimeric G protein. When occupied by ligand, the pheromone receptors activate the pheromone-responsive Ga protein (encoded by GPA1) via guanine nucleotide exchange and the concomitant dissociation of Ga?GTP from the Gbg dimer (encoded by STE4 and STE18). The signal is then transmitted by Gbg to a mitogen?activated protein (MAP) kinase cascade. The MAP kinase module consists of Ste11 (the MEKK), Ste7 (the MEK), and Fus3 (the MAPK). In addition to stimulating the mating signal, Gbg serves as a positional cue. It directs the dual function protein, Far1, to the growth site. Far1 serves as a scaffold. It brings together elements that stimulate polarization of the actin cytoskeleton. Under physiological conditions, the Gbg-Far1 complex is presumed to assemble in the region of the cell surface that experiences the highest concentration of pheromone, and to mark this area for growth. Thus, the cell orients its growth toward the source of pheromone. This is called chemotropism. Recent results implicate the pheromone-responsive Ga protein, Gpa1, in control of signal-induced polarization and nuclear movement. In cells responding to pheromone, Gpa1 interacts directly with the mating-specific MAP kinase, Fus3, and with Kar3, a kinesin-like protein that is essential for nuclear movement during mating. Disruption of the Gpa1-Fus3 interaction confers defects in chemotropism and nuclear migration. The goals of this investigation are to determine which chemotropic factors depend on the recruitment of Fus3 by Gpa1, and to determine how Gpa1 affects nuclear movement during mating. In the work funded by NSF, the following hypothesis will be tested: Gpa1 co-localizes Fus3 and Kar3 so that Fus3 can activate Kar3. Because there is no precedent for regulation of kinesins by Ga proteins or MAP kinases, and because it is not known how kinesins are activated, this work promises to be of great value to those studying microtubule-associated motor proteins. The broader impact of this investigation is that it will enhance our understanding of chemotropism and nuclear movement, two fundamental aspects of cellular function. Students at all levels will be involved in this research.