Fang Hao-Ming, Wang Yue
Institute of Molecular and Cell Biology, ASTAR Biomedical Sciences Institutes, Proteos, 61 Biopolis Drive, Singapore 138673, Singapore.
Mol Microbiol. 2006 Jul;61(2):484-96. doi: 10.1111/j.1365-2958.2006.05248.x.
Many Ras GTPases activate their effectors through binding at a conserved Ras association (RA) domain. An example is the activation of the budding yeast adenylate cyclase Cyr1 by Ras1 and Ras2. Candida albicans Ras1 is speculated to similarly activate Cdc35, the orthologue of Cyr1, for hyphal development. Here, we have investigated whether the RA domain mediates Ras1-Cdc35 interaction and how this interaction regulates cAMP levels and morphogenesis. Yeast two-hybrid assays suggested that Ras1 interacts only with the RA but not any other identifiable domains of Cdc35. The Ras1-RA interaction was further confirmed by in vitro binding assays of purified RA domain and Ras1 and by co-immunoprecipitation of Ras1 and Cdc35 from cell lysates. Substituting Ala for the conserved residue K(338) or L(349) in the RA domain or deleting the RA domain abolished the Ras1-RA or Ras1-Cdc35 interactions. cdc35 mutants with the RA domain deleted or carrying K388A or L349A mutation exhibited rather normal yeast growth but were completely defective in hyphal morphogenesis. Further, the mutants contained nearly wild-type levels of cAMP during yeast growth but were unable to increase it upon hyphal induction. These results suggest an essential role for the RA-mediated Ras1-Cdc35 interaction in raising cellular cAMP levels for hyphal morphogenesis.
许多Ras GTP酶通过与保守的Ras关联（RA）结构域结合来激活其效应器。一个例子是芽殖酵母腺苷酸环化酶Cyr1被Ras1和Ras2激活。推测白色念珠菌Ras1同样通过激活Cyr1的直系同源物Cdc35来促进菌丝发育。在这里，我们研究了RA结构域是否介导Ras1与Cdc35的相互作用，以及这种相互作用如何调节cAMP水平和形态发生。酵母双杂交试验表明，Ras1仅与Cdc35的RA结构域相互作用，而不与其他任何可识别的结构域相互作用。纯化的RA结构域与Ras1的体外结合试验以及从细胞裂解物中对Ras1和Cdc35进行的免疫共沉淀进一步证实了Ras1与RA结构域的相互作用。将RA结构域中保守残基K（338）或L（349）替换为丙氨酸或删除RA结构域会消除Ras1与RA结构域或Ras1与Cdc35的相互作用。缺失RA结构域或携带K388A或L349A突变的cdc35突变体在酵母生长过程中表现出相当正常的生长，但在菌丝形态发生方面完全缺陷。此外，这些突变体在酵母生长期间含有接近野生型水平的cAMP，但在菌丝诱导时无法增加cAMP水平。这些结果表明，RA介导的Ras1与Cdc35的相互作用在提高细胞cAMP水平以促进菌丝形态发生中起着至关重要的作用。
