Piispanen Amy E, Bonnefoi Ophelie, Carden Sarah, Deveau Aurelie, Bassilana Martine, Hogan Deborah A
Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, NH 03755, USA.
Eukaryot Cell. 2011 Nov;10(11):1473-84. doi: 10.1128/EC.05153-11. Epub 2011 Sep 9.
Many Ras GTPases localize to membranes via C-terminal farnesylation and palmitoylation, and localization regulates function. In Candida albicans, a fungal pathogen of humans, Ras1 links environmental cues to morphogenesis. Here, we report the localization and membrane dynamics of Ras1, and we characterize the roles of conserved C-terminal cysteine residues, C287 and C288, which are predicted sites of palmitoylation and farnesylation, respectively. GFP-Ras1 is localized uniformly to plasma membranes in both yeast and hyphae, yet Ras1 plasma membrane mobility was reduced in hyphae compared to that in yeast. Ras1-C288S was mislocalized to the cytoplasm and could not support hyphal development. Ras1-C287S was present primarily on endomembranes, and strains expressing ras1-C287S were delayed or defective in hyphal induction depending on the medium used. Cells bearing constitutively activated Ras1-C287S or Ras1-C288S, due to a G13V substitution, showed increased filamentation, suggesting that lipid modifications are differentially important for Ras1 activation and effector interactions. The C. albicans autoregulatory molecule, farnesol, inhibits Ras1 signaling through adenylate cyclase and bears structural similarities to the farnesyl molecule that modifies Ras1. At lower concentrations of farnesol, hyphal growth was inhibited but Ras1 plasma membrane association was not altered; higher concentrations of farnesol led to mislocalization of Ras1 and another G protein, Rac1. Furthermore, farnesol inhibited hyphal growth mediated by cytosolic Ras1-C288SG13V, suggesting that farnesol does not act through mechanisms that depend on Ras1 farnesylation. Our findings imply that Ras1 is farnesylated and palmitoylated, and that the Ras1 stimulation of adenylate cyclase-dependent phenotypes can occur in the absence of these lipid modifications.
许多Ras GTP酶通过C端法尼基化和棕榈酰化定位于细胞膜，而定位于细胞膜又调控其功能。在人类真菌病原体白色念珠菌中，Ras1将环境信号与形态发生联系起来。在此，我们报告了Ras1的定位及膜动力学，并对保守的C端半胱氨酸残基C287和C288的作用进行了表征，预测它们分别是棕榈酰化和法尼基化位点。GFP-Ras1在酵母和菌丝中均均匀定位于质膜，但与酵母相比，Ras1在菌丝中的质膜流动性降低。Ras1-C288S错定位于细胞质，无法支持菌丝发育。Ras1-C287S主要存在于内膜上，根据所用培养基的不同，表达ras1-C287S的菌株在菌丝诱导方面会延迟或存在缺陷。由于G13V替换而携带组成型激活的Ras1-C287S或Ras1-C288S的细胞显示出菌丝形成增加，这表明脂质修饰对于Ras1激活和效应物相互作用具有不同的重要性。白色念珠菌的自调节分子法尼醇通过腺苷酸环化酶抑制Ras1信号传导，并且与修饰Ras1的法尼基分子具有结构相似性。在较低浓度的法尼醇下，菌丝生长受到抑制，但Ras1与质膜的结合未改变；较高浓度的法尼醇导致Ras1和另一种G蛋白Rac1的定位错误。此外，法尼醇抑制由胞质Ras1-C288SG13V介导的菌丝生长，这表明法尼醇并非通过依赖Ras1法尼基化的机制起作用。我们的研究结果表明，Ras1被法尼基化和棕榈酰化，并且在没有这些脂质修饰的情况下，Ras1对腺苷酸环化酶依赖性表型的刺激也可能发生。
