Leptomycin B targets a regulatory cascade of crm1, a fission yeast nuclear protein, involved in control of higher order chromosome structure and gene expression.
The molecular action of leptomycin B (LMB), an agent inducing arrest of the eukaryotic cell cycle at G1 and G2 phases, was investigated by analyzing an LMB resistance gene of Schizosaccharomyces pombe. A genomic library of an LMB-resistant mutant was screened for LMB resistance, and a DNA fragment containing an open reading frame (ORF) of 1078 amino acids was cloned on a multicopy vector. The plasmid was found to confer drug resistance specifically to LMB. Nucleotide sequencing revealed that the ORF was a mutant gene for the essential nuclear protein crm1, which had been reported to complement a cold-sensitive mutation causing deformed nuclear morphology. The gene product named crm1-N1 had two amino acid replacements (Gly-503 to Asp and Met-546 to Ile). Two allelic mutants of crm1 (crm1-809 and crm1-119) were found to be hypersensitive and resistant, respectively, to LMB. Nuclear morphology of the cold-sensitive crm1-809 mutant at the restrictive temperature was almost the same as that of the wild-type cells treated with LMB. Furthermore, a low concentration of LMB induced the intracellular accumulation of a 25-kDa protein in the wild-type cells, which was immunologically identical to the protein accumulating in the crm1-809 mutant cells. These results strongly suggest that LMB primarily inhibits the function of the crm1 gene which is required for maintaining higher order chromosome structures, correct gene expression, and cell growth in the fission yeast.
Nishi
K
K
Department of Agricultural Chemistry Faculty of Agriculture, University of Tokyo, Japan.
Yoshida
M
M
Fujiwara
D
D
Nishikawa
M
M
Horinouchi
S
S
Beppu
T
T
D000470:Alkaloids / Q000494:pharmacology; D000483:Alleles; D000935:Antifungal Agents / Q000494:pharmacology*; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D003513:Cycloheximide / Q000494:pharmacology; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D005231:Fatty Acids, Unsaturated / Q000302:isolation & purification / Q000494:pharmacology; D005656:Fungal Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005800:Genes, Fungal; D028884:Karyopherins; D009687:Nuclear Proteins / Q000378:metabolism*; D016366:Open Reading Frames; D017354:Point Mutation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D018160:Receptors, Cytoplasmic and Nuclear; D015183:Restriction Mapping; D012568:Schizosaccharomyces / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D019311:Staurosporine; D016385:TATA Box; D014157:Transcription Factors / Q000378:metabolism*; D014634:Valinomycin / Q000494:pharmacology; D000097606:Exportin 1 Protein
D000470:Alkaloids; D000935:Antifungal Agents; D003513:Cycloheximide; D004271:DNA, Fungal; D005231:Fatty Acids, Unsaturated; D005656:Fungal Proteins; D028884:Karyopherins; D009687:Nuclear Proteins; D011493:Protein Kinase C; D018160:Receptors, Cytoplasmic and Nuclear; D019311:Staurosporine; D014157:Transcription Factors; D014634:Valinomycin; D000097606:Exportin 1 Protein; C038753:leptomycin B