Fytlovich S, Gervais M, Agrimonti C, Guiard B
Centre de Génétique Moléculaire, Laboratoire propre du Centre National de la Recherche Scientifique associé à l'Université Pierre et Marie Curie, Gif-sur-Yvette, France.
EMBO J. 1993 Mar;12(3):1209-18. doi: 10.1002/j.1460-2075.1993.tb05762.x.
Previously, it was shown that the CYP1(HAP1) gene product mediates the transcription of several oxygen-regulated genes through a metabolic co-effector, heme, in the yeast Saccharomyces cerevisiae. This study investigates the overproduction of the CYP1 protein when the CYP1(HAP1) gene is placed under the control of the GAL10-CYC1 hybrid promoter (either at the locus of the CYP1(HAP1) gene or cloned in a high-copy-number plasmid). In these conditions, the CYP1 protein is detected by Western blot analysis and has a molecular mass in agreement with the open reading frame sequence. Band-shift experiments show that the CYP1(HAP1) protein is able to interact specifically with its target sequences in vitro without addition of hemin, and forms a large complex with one or several unidentified factors denoted as X. Addition of hemin allows the formation of a new complex which has a lower molecular mass. The internal deletion of the seven repeated amino acid sequences containing the KCPVDH motif in the CYP1(HAP1) protein modifies the heme responsiveness phenomenon observed in vitro in the band-shift experiments and in vivo in the transcription of the CYB2, CYC1, CYP3(CYC7) and ERG11 genes. On the basis of these data, we propose a new model for heme-induced activation of the CYP1 protein.
此前的研究表明，在酿酒酵母中，CYP1（HAP1）基因产物通过代谢共效应物血红素介导几种氧调节基因的转录。本研究调查了将CYP1（HAP1）基因置于GAL10-CYC1杂合启动子控制下（要么在CYP1（HAP1）基因位点，要么克隆到高拷贝数质粒中）时CYP1蛋白的过量表达情况。在这些条件下，通过蛋白质免疫印迹分析检测到CYP1蛋白，其分子量与开放阅读框序列一致。凝胶迁移实验表明，CYP1（HAP1）蛋白在不添加血红素的情况下能够在体外与其靶序列特异性相互作用，并与一种或几种未鉴定的因子X形成大的复合物。添加血红素后可形成一种分子量较低的新复合物。CYP1（HAP1）蛋白中包含KCPVDH基序的七个重复氨基酸序列的内部缺失改变了在凝胶迁移实验中体外观察到的以及在CYB2、CYC1、CYP3（CYC7）和ERG11基因转录中体内观察到的血红素反应现象。基于这些数据，我们提出了一种血红素诱导CYP1蛋白激活的新模型。
