Vallier Laura G, Segall Jeffrey E, Snyder Michael
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA.
Cell Motil Cytoskeleton. 2002 Dec;53(4):251-66. doi: 10.1002/cm.10073.
Successful mating of MATa Saccharomyces cerevisiae cells is dependent on Ste2p, the alpha-factor receptor. Besides receiving the pheromone signal and transducing it through the G-protein coupled MAP kinase pathway, Ste2p is active in the establishment and orientation of the mating projection. We investigated the role of the carboxyl terminus of the receptor in mating projection formation and orientation using a spatial gradient assay. Cells carrying the ste2-T326 mutation, truncating 105 of the 135 amino acids in the receptor tail including a motif necessary for its ligand-mediated internalization, display slow onset of projection formation, abnormal shmoo morphology, and reduced ability to orient the mating projection toward a pheromone source. This reduction was due to the increased loss of mating projection orientation in a pheromone gradient. Cells with a mutated endocytosis motif were defective in reorientation in a pheromone gradient. ste2-Delta296 cells, which carry a complete truncation of the Ste2p tail, exhibit a severe defect in projection formation, and those projections that do form are unable to orient in a pheromone gradient. These results suggest a complex role for the Ste2p carboxy-terminal tail in the formation, orientation, and directional adjustment of the mating projection, and that endocytosis of the receptor is important for this process. In addition, mutations in RSR1/BUD1 and SPA2, genes necessary for budding polarity, exhibited little or no defect in formation or orientation of mating projections. We conclude that mating projection orientation depends upon the carboxyl terminus of the pheromone receptor and not the directional machinery used in budding.
MATa 型酿酒酵母细胞的成功交配依赖于α-因子受体Ste2p。除了接收信息素信号并通过G蛋白偶联的丝裂原活化蛋白激酶途径进行转导外，Ste2p在交配突起的形成和定向中也发挥作用。我们使用空间梯度试验研究了受体羧基末端在交配突起形成和定向中的作用。携带ste2-T326突变的细胞，其受体尾部135个氨基酸中的105个被截断，包括其配体介导的内化所必需的基序，表现出突起形成起始缓慢、异常的shmoo形态，以及将交配突起朝向信息素源定向的能力降低。这种降低是由于在信息素梯度中交配突起定向的丧失增加所致。具有突变的内吞基序的细胞在信息素梯度中的重新定向存在缺陷。携带Ste2p尾巴完全截断的ste2-Δ296细胞在突起形成方面存在严重缺陷，并且那些形成的突起无法在信息素梯度中定向。这些结果表明Ste2p羧基末端尾巴在交配突起的形成、定向和方向调整中具有复杂的作用，并且受体的内吞作用对这一过程很重要。此外，芽殖极性所需基因RSR1/BUD1和SPA2中的突变在交配突起的形成或定向中几乎没有缺陷。我们得出结论，交配突起的定向取决于信息素受体的羧基末端，而不是芽殖中使用的定向机制。
