Far3 and five interacting proteins prevent premature recovery from pheromone arrest in the budding yeast Saccharomyces cerevisiae
Far3 and five interacting proteins prevent premature recovery from pheromone arrest in the budding yeast Saccharomyces cerevisiae
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DOI：
10.1128/mcb.23.5.1750-1763.2003
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发表时间：
2003-03-01
影响因子：
5.3
通讯作者：
Sprague, GF
中科院分区：
文献类型：
--
作者：
Kemp, HA;Sprague, GF
在出芽酵母中，可扩散的交配信息素启动一个信号通路，最终产生包括细胞周期停滞在内的若干反应。在信息素反应和细胞周期之间的相互作用所必需的基因中，只有少数几个已被确定，其中包括FAR1和FAR3；在这些基因中，只有FAR1得到了广泛的研究。为了了解Far3作用的机制，我们使用双杂交方法来鉴定相互作用的蛋白质。我们鉴定了5个以前未被研究的开放阅读框，分别命名为FAR7、FAR8、FAR9、FAR10和FAR11，当这些基因被破坏时，会导致类似far3的信息素停滞缺陷。通过双杂交和免疫共沉淀分析，我们发现所有6种Far蛋白都相互作用。此外，速率沉降实验表明Far3以及Far7到Far11形成一个复合物。六重far3 far7 - far11突变体的表型并不比任何单个突变体更严重。因此，FAR3以及FAR7到FAR11都参与了导致G₁停滞的同一通路。这些突变体最初会因信息素而停滞，但在10小时后会重新出芽。在这些条件下，野生型细胞即使几天后也不会重新出芽，而far1突变体细胞在1小时内就会重新出芽。我们得出结论，依赖于FAR3的停滞通路在功能上不同于利用FAR1的通路。
In budding yeast, diffusible mating pheromones initiate a signaling pathway that culminates in several responses, including cell cycle arrest. Only a handful of genes required for the interface between pheromone response and the cell cycle have been identified, among them FAR1 and FAR3; of these, only FAR] has been extensively characterized. In an effort to learn about the mechanism by which Far3 acts, we used the two-hybrid method to identify interacting proteins. We identified five previously uncharacterized open reading frames, dubbed FAR7, FAR8, FAR9, FAR10, and FAR11, that cause a far3-like pheromone arrest defect when disrupted. Using two-hybrid and coimmunoprecipitation analysis, we found that all six Far proteins interact with each other. Moreover, velocity sedimentation experiments suggest that Far3 and Far7 to Far11 form a complex. The phenotype of a sextuple far3far7-far11 mutant is no more severe than any single mutant. Thus, FAR3 and FAR7 to FAR11 all participate in the same pathway leading to G, arrest. These mutants initially arrest in response to pheromone but resume budding after 10 h. Under these conditions, wild-type cells fail to resume budding even after several days whereas far1 mutant cells resume budding within 1 h. We conclude that the FAR3-dependent arrest pathway is functionally distinct from that which employs FAR1.