RESUMO
The cellular target of leptomycin B (LMB), a nuclear export inhibitor, has been identified as CRM1 (exportin 1), an evolutionarily conserved receptor for the nuclear export signal of proteins. However, the mechanism by which LMB inhibits CRM1 still remains unclear. CRM1 in a Schizosaccharomyces pombe mutant showing extremely high resistance to LMB had a single amino acid replacement at Cys-529 with Ser. The mutant gene, named crm1-K1, conferred LMB resistance on wild-type S. pombe, and Crm1-K1 no longer bound biotinylated LMB. (1)H NMR analysis showed that LMB bound N-acetyl-L-cysteine methyl ester through a Michael-type addition, consistent with the idea that LMB binds covalently via its alpha, beta-unsaturated delta-lactone to the sulfhydryl group of Cys-529. When HeLa cells were cultured with biotinylated LMB, the only cellular protein bound covalently was CRM1. Inhibition by N-ethylmaleimide (NEM), an alkylating agent, of CRM1-mediated nuclear export probably was caused by covalent binding of the electrophilic structure in NEM to the sulfhydryl group of Cys-529, because the crm1-K1 mutant showed the normal rate for the export of Rev nuclear export signal-bearing proteins in the presence of not only LMB but also NEM. These results show that the single cysteine residue determines LMB sensitivity and is selectively alkylated by LMB, leading to CRM1 inactivation.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Cisteína , Carioferinas , Receptores Citoplasmáticos e Nucleares , Schizosaccharomyces/fisiologia , Sequência de Aminoácidos , Sítios de Ligação , Biotinilação , Proteínas de Transporte/genética , Sequência Conservada , Primers do DNA , Resistência Microbiana a Medicamentos/genética , Ácidos Graxos Insaturados/farmacologia , Genes Fúngicos , Células HeLa , Humanos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Schizosaccharomyces/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Moldes Genéticos , Transcrição Gênica , Proteína Exportina 1RESUMO
Leptomycin B (LMB) is a Streptomyces metabolite that inhibits nuclear export of the human immunodeficiency virus type 1 regulatory protein Rev at low nanomolar concentrations. Recently, LMB was shown to inhibit the function of CRM1, a receptor for the nuclear export signal (NES). Here we show evidence that LMB binds directly to CRM1 and that CRM1 is essential for NES-dependent nuclear export of proteins in both yeast and mammalian cells. Binding experiments with a biotinylated derivative of LMB and a HeLa cell extract led to identifying CRM1 as a major protein that bound to the LMB derivative. Microinjection of a purified anti-human CRM1 antibody into the mammalian nucleus specifically inhibited nuclear export of NES-containing proteins, as did LMB. Consistent with this, CRM1 was found to interact with NES, when assayed with immobilized NES and HeLa cell extracts. This association was disrupted by adding LMB or purified anti-human CRM1 antibody. The inhibition of CRM1 by LMB was also observed in fission yeast. The fission yeast crm1 mutant was defective in the nuclear export of NES-fused proteins, but not in the import of nuclear localization signal (NLS)-fused proteins. Interestingly, a protein containing both NES and NLS, which is expected to shuttle between nucleus and cytoplasm, was highly accumulated in the nucleus of the crm1 mutant cells or of cells treated with LMB. These results strongly suggest that CRM1 is the target of LMB and is an essential factor for nuclear export of proteins in eukaryotes.
