Identification of Saccharomyces cerevisiae isoleucyl-tRNA synthetase as a target of the G1-specific inhibitor reveromycin A
Identification of Saccharomyces cerevisiae isoleucyl-tRNA synthetase as a target of the G1-specific inhibitor reveromycin A
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DOI：
10.1074/jbc.m203827200
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发表时间：
2002-08-09
影响因子：
4.8
通讯作者：
Miyakawa, T
中科院分区：
文献类型：
--
作者：
Miyamoto, Y;Machida, K;Miyakawa, T
为探讨还原霉素A（reveromycin A，RM-A）的作用机制，从缺失非特异性多药耐药相关基因的酿酒酵母（Saccharomyces cerevisiae）中筛选出一株对RM-A具有特异性抗性的G（1）抑制剂--RM-A显性突变株。鉴定了负责抗性的突变基因（YRR 2 -1）是编码tRNA（Ile）合成酶（MeRS）的ILS 1基因的等位基因。在野生型细胞提取物中检测到的HeRS活性对RM-A高度敏感（IC 50 = 8 ng/ml），而非其他几种氨酰-tRNA合成酶。YRR 2 -1突变体的IleRS活性比野生型的IleRS活性高4倍。突变IleRS（N660 D），附近的KMSKS共识序列中常见的I类氨酰转移酶，被认为是负责RM-A的阻力。此外，ILS 1基因从高拷贝质粒的过表达赋予RM-A抗性。这些结果表明，IleRS是RM-A在体内的靶点。GCN 2基因的缺陷导致RM-A抗性下降。RM-A抑制IleRS导致ILS 1基因通过Gcn 2-Gcn 4一般氨基酸控制途径的转录激活，并且这种自动调节似乎有助于RM-A抗性。
To dissect the action mechanism of reveromycin A (RM-A), a G(1)-specific inhibitor, a Saccharomyces cerevisiae dominant mutant specifically resistant to RM-A, was isolated from a strain in which the genes implicated in nonspecific multidrug resistance had been deleted. The mutant gene (YRR2-1) responsible for the resistance was identified as an allele of the ILS1 gene encoding tRNA(Ile) synthetase (MeRS). The activity of HeRS, but not several other aminoacyl-tRNA synthetases examined in wild type cell extract, was highly sensitive to RM-A (IC50 = 8 ng/ml). The IleRS activity of the YRR2-1 mutant was 4-fold more resistant to the inhibitor compared with that of wild type. The mutation IleRS(N660D), near the KMSKS consensus sequence commonly found in the class I aminoacyl transferases, was found to be responsible for RM-A resistance. Moreover, overexpression of the ILS1 gene from a high-copy plasmid conferred RM-A resistance. These results indicated that IleRS is a target of RM-A in vivo. A defect of the GCN2 gene led to decreased RM-A resistance. IleRS inhibition by RM-A led to transcriptional activation of the ILS1 gene via the Gcn2-Gcn4 general amino acid control pathway, and this autoregulation seemed to contribute to RM-A resistance.