Department of Resource and Environmental Science, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200240, China.
Microbiol Res. 2012 Oct 12;167(9):536-43. doi: 10.1016/j.micres.2012.02.002. Epub 2012 Mar 6.
The Mac1 protein was a transcriptional activator that sensed very low concentration of copper and regulated the copper transport in Saccharomyces cerevisiae. Here, we cloned a gene from Trichoderma reesei named Tmac1, whose deduced amino acid sequence showed 29% identical to Mac1p. Furthermore, two Cys-His repeats metal binding motifs of Tmac1p, one in the 354-369C terminus and one in the 475-490C terminus were also present in Mac1p. A deletion mutant of Tmac1 was hypersensitive to the copper starvation and showed poor growth. Subsequently, the function was recovered by the gene complementation experiment. Furthermore, the Tmac1 gene fully complemented growth defects of yeast ΔMac1 mutant. The expression of Tmac1p was activated at low concentration of copper and depressed when the concentration of copper excess 1mM. Furthermore, the fluorescence intensity enhanced at copper starvation and decreased under copper excess by fusion the eGFP to the Tmac1p. It proved that the expression of Mac1p was exactly regulated by copper concentration, because eGFP and Mac1p were expressed under the control of the same one promoter. We also cloned a gene named Tctr3 with bioinformatics. With a series of experiments, we proved it was the target gene of Tmac1. To sum up, Tmac1 may encode a transcriptional activator regulated high-affinity copper transport in T. reesei.
Mac1 蛋白是一种转录激活因子，能够感应极低浓度的铜，并调节酿酒酵母中的铜转运。在这里，我们从里氏木霉中克隆了一个名为 Tmac1 的基因，其推导的氨基酸序列与 Mac1p 有 29%的相同性。此外，Tmac1p 中存在两个 Cys-His 重复金属结合基序，一个在 354-369C 末端，一个在 475-490C 末端，与 Mac1p 中的基序相同。Tmac1 的缺失突变体对铜饥饿敏感，生长不良。随后，通过基因互补实验恢复了功能。此外，Tmac1 基因完全弥补了酵母 ΔMac1 突变体的生长缺陷。Tmac1p 的表达在低浓度铜时被激活，而在铜过量 1mM 时被抑制。此外，通过将 eGFP 融合到 Tmac1p 上，在铜饥饿时荧光强度增强，在铜过量时荧光强度降低。这证明了 Mac1p 的表达确实受到铜浓度的调控，因为 eGFP 和 Mac1p 是在同一个启动子的控制下表达的。我们还通过生物信息学克隆了一个名为 Tctr3 的基因。通过一系列实验，我们证明它是 Tmac1 的靶基因。总之，Tmac1 可能编码一种转录激活因子，调节里氏木霉中的高亲和力铜转运。
