RA domain-mediated interaction of Cdc35 with Ras1 is essential for increasing cellular cAMP level for Candida albicans hyphal development
RA domain-mediated interaction of Cdc35 with Ras1 is essential for increasing cellular cAMP level for Candida albicans hyphal development
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DOI：
10.1111/j.1365-2958.2006.05248.x
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发表时间：
2006-07-01
影响因子：
3.6
通讯作者：
Wang, Yue
中科院分区：
文献类型：
--
作者：
Fang, Hao-Ming;Wang, Yue
许多Ras GTP酶通过与保守的Ras缔合（RA）结构域结合来激活其效应子。一个例子是Ras 1和Ras 2对芽殖酵母腺苷酸环化酶Cyr 1的激活。白色念珠菌Ras 1被推测为类似地激活Cdc 35，Cyr 1的直系同源物，用于菌丝发育。在这里，我们研究了RA域是否介导Ras 1-Cdc 35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，Ras 1只与RA相互作用，但不与Cdc 35的任何其他可识别的结构域。Ras 1-RA相互作用进一步证实了纯化的RA结构域和Ras 1的体外结合试验，并从细胞裂解物的Ras 1和Cdc 35的免疫共沉淀。用Ala取代RA结构域中的保守残基K-338或L-349或缺失RA结构域可消除Ras 1-RA或Ras 1-Cdc 35相互作用。缺失RA结构域或携带K388 A或L349 A突变的cdc 35突变体表现出相当正常的酵母生长，但菌丝形态发生完全缺陷。此外，突变体在酵母生长过程中含有接近野生型水平的cAMP，但在菌丝诱导后不能增加cAMP。这些结果表明RA介导的Ras 1-Cdc 35相互作用在提高细胞cAMP水平的菌丝形态发生中起重要作用。
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.