Zhang Anda, Petrov Kostadin O, Hyun Emily R, Liu Zhongle, Gerber Scott A, Myers Lawrence C
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire, USA.
Eukaryot Cell. 2012 Jul;11(7):874-84. doi: 10.1128/EC.00095-12. Epub 2012 May 4.
The amplification of the TLO (for telomere-associated) genes in Candida albicans, compared to its less pathogenic, close relative Candida dubliniensis, suggests a role in virulence. Little, however, is known about the function of the Tlo proteins. We have purified the Mediator coactivator complex from C. albicans (caMediator) and found that Tlo proteins are a stoichiometric component of caMediator. Many members of the Tlo family are expressed, and each is a unique member of caMediator. Protein expression analysis of individual Tlo proteins, as well as the purification of tagged Tlo proteins, demonstrate that there is a large free population of Tlo proteins in addition to the Mediator-associated population. Coexpression and copurification of Tloα12 and caMed3 in Escherichia coli established a direct physical interaction between the two proteins. We have also made a C. albicans med3Δ/Δ strain and purified an intact Mediator from this strain. The analysis of the composition of the med3Δ Mediator shows that it lacks a Tlo subunit. Regarding Mediator function, the med3Δ/Δ strain serves as a substitute for the difficult-to-make tloΔ/Δ C. albicans strain. A potential role of the TLO and MED3 genes in virulence is supported by the inability of the med3Δ/Δ strain to form normal germ tubes. This study of caMediator structure provides initial clues to the mechanism of action of the Tlo genes and a platform for further mechanistic studies of caMediator's involvement in gene regulatory patterns that underlie pathogenesis.
与致病性较低的近缘种白假丝酵母相比，白色念珠菌中TLO（端粒相关）基因的扩增表明其在毒力方面发挥作用。然而，人们对Tlo蛋白的功能知之甚少。我们从白色念珠菌中纯化了中介体共激活复合物（caMediator），发现Tlo蛋白是caMediator的化学计量成分。Tlo家族的许多成员都有表达，且每个成员都是caMediator的独特成员。对单个Tlo蛋白的蛋白质表达分析以及带标签的Tlo蛋白的纯化表明，除了与中介体相关的群体外，还有大量游离的Tlo蛋白群体。在大肠杆菌中共表达和共纯化Tloα12和caMed3，证实了这两种蛋白之间存在直接的物理相互作用。我们还构建了白色念珠菌med3Δ/Δ菌株，并从该菌株中纯化了完整的中介体。对med3Δ中介体组成的分析表明，它缺少一个Tlo亚基。关于中介体功能，med3Δ/Δ菌株可替代难以构建的白色念珠菌tloΔ/Δ菌株。med3Δ/Δ菌株无法形成正常的芽管，这支持了TLO和MED3基因在毒力方面的潜在作用。这项对caMediator结构的研究为Tlo基因的作用机制提供了初步线索，并为进一步研究caMediator参与发病机制基础的基因调控模式的机制研究提供了一个平台。
