Identification of Saccharomyces cerevisiae isoleucyl-tRNA synthetase as a target of the G1-specific inhibitor Reveromycin A.
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 (IleRS). The activity of IleRS, but not several other aminoacyl-tRNA synthetases examined in wild type cell extract, was highly sensitive to RM-A (IC(50) = 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.
Miyamoto
Yuji
Y
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima 739-8530, Japan.
Machida
Kiyotaka
K
Mizunuma
Masaki
M
Emoto
Yuji
Y
Sato
Naomi
N
Miyahara
Kohji
K
Hirata
Dai
D
Usui
Takeo
T
Takahashi
Hidetoshi
H
Osada
Hiroyuki
H
Miyakawa
Tokichi
T
D002453:Cell Cycle / Q000187:drug effects; D002469:Cell Separation; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D018432:Drug Resistance, Multiple; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011714:Pyrans / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013141:Spiro Compounds / Q000494:pharmacology*; D013696:Temperature; D013997:Time Factors; D014158:Transcription, Genetic
D011714:Pyrans; D013141:Spiro Compounds; C068098:reveromycin A; D008715:Methionine; D007533:Isoleucine-tRNA Ligase