Sahu Pushpendra Kumar, Chauhan Sakshi, Tomar Raghuvir Singh
Laboratory of Chromatin Biology, Department of Biological Sciences , Indian Institute of Science Education and Research Bhopal , Bhopal 462066 , Madhya Pradesh , India.
Biochemistry. 2019 Apr 2;58(13):1799-1809. doi: 10.1021/acs.biochem.8b01277. Epub 2019 Mar 12.
Crg1 is an S-adenosylmethionine (SAM)-dependent methyltransferase required for cantharidin resistance in yeast. Crg1 has a well-characterized methyltransferase domain that inactivates cantharidin by methylation. However, the remaining part of the Crg1 protein is yet to be functionally characterized. In this study, we identified an essential role of the N-terminus of Crg1 in methyltransferase activity and cantharidin resistance. Yeast cells lacking 41 residues of the N-terminus of Crg1 ( crg1ΔN) showed hypersensitivity to cantharidin as same as the null mutant, crg1. The mass spectrometry-based biochemical enzyme assay revealed a loss of methyltransferase activity in Crg1ΔN, which justifies the loss of cantharidin resistance, as well. The subcellular distribution of Crg1ΔN-daGFP showed cytoplasmic aggregates, whereas wild-type Crg1-daGFP was distributed normally in the cytoplasm. Interestingly, the Crg1-methyltransferase domain point mutants (D44A, D67A, and E105A/D108A) also showed the same cytoplasmic aggregates as Crg1ΔN-daGFP. In silico prediction of the tertiary structures of these mutants indicated an altered protein conformation. Altogether, these observations suggest that the N-terminal truncation, as well as the point mutations in the methyltransferase domain, alters the native folding of Crg1 methyltransferase, resulting in a loss of enzyme activity. Furthermore, the crg1ΔN mutant showed the same phenotypes as the crg1 null mutant in the presence of cantharidin, i.e., lethal cell growth, PE auxotrophy, temperature sensitivity, endoplasmic reticulum stress, GPI anchor missorting, and cell wall damage. Overall, this study identifies an essential role of the N-terminus of Crg1 in methyltransferase activity and cantharidin resistance.
Crg1是酵母中抗斑蝥素所必需的一种依赖S-腺苷甲硫氨酸（SAM）的甲基转移酶。Crg有一个特征明确的甲基转移酶结构域，该结构域通过甲基化使斑蝥素失活。然而，Crg1蛋白的其余部分尚未进行功能表征。在本研究中，我们确定了Crg1的N端在甲基转移酶活性和抗斑蝥素方面的重要作用。缺乏Crg1 N端41个残基的酵母细胞（crg1ΔN）对斑蝥素表现出与缺失突变体crg1相同的超敏感性。基于质谱的生化酶分析显示Crg1ΔN中甲基转移酶活性丧失，这也解释了其抗斑蝥素能力的丧失。Crg1ΔN-daGFP的亚细胞分布显示为细胞质聚集体，而野生型Crg1-daGFP正常分布于细胞质中。有趣的是，Crg1甲基转移酶结构域点突变体（D44A、D67A和E105A/D108A）也表现出与Crg1ΔN-daGFP相同的细胞质聚集体。对这些突变体三级结构的计算机预测表明蛋白质构象发生了改变。总之，这些观察结果表明，N端截短以及甲基转移酶结构域中的点突变会改变Crg1甲基转移酶的天然折叠，导致酶活性丧失。此外，在存在斑蝥素的情况下，crg1ΔN突变体表现出与crg1缺失突变体相同的表型，即致死性细胞生长、PE营养缺陷、温度敏感性、内质网应激、GPI锚定分选错误和细胞壁损伤。总体而言，本研究确定了Crg1的N端在甲基转移酶活性和抗斑蝥素方面的重要作用。
