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 Michaeltype addition, consistent with the idea that LMB binds covalently via its ␣,␤-unsaturated ␦-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.Many cellular proteins either reside in the nucleus or shuttle between the nucleus and the cytoplasm by energy-dependent transport across the nuclear envelope. Specific sequences within a protein contain the information necessary for the nucleocytoplasmic transport: most nuclear proteins have nuclear localization sequences (NLS) rich in basic amino acids, whereas others carry short nuclear export sequences (NES) rich in leucine (1, 2). CRM1͞exportin 1 was shown to be a receptor for the NES in both lower and higher eukaryotes (3-6). Genetic alterations in the CRM1 locus caused a defect in nuclear export of NES-bearing proteins in yeast (3,5,7,8). Nuclear microinjection of a CRM1-specific antibody that prevents the in vitro NES binding inhibited in vivo protein nuclear export in mammalian cells (9). Thus, the NESmediated nuclear export of proteins is a universal and conserved mechanism by which subcellular localization of proteins is controlled in cells.CRM1 originally was identified as a protein essential for maintaining chromosome structure in the fission yeast Schizosaccharomyces pombe (10). The functional homologues that complement the fission yeast crm1 mutation were cloned from the budding yeast Saccharomyces cerevisiae (11) and from human cells (8,12). We showed previously that a mutation (crm1-N1) of S. pombe crm1 ϩ conferred resistance to leptomycin B (LMB) (13), which had been discovered as a potent antifungal antibiotic blocking the eukaryotic cell cycle (14, 15). In contrast, the cold-sensitive crm1-809 mutant strain was hypersensitive to LMB. Furthermore, morphological and biochemical phenotypes of crm1-809 mutant cells at nonpermissive temperature ...

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Leptomycin B is the first low molecular weight inhibitor of nuclear export to be identified. Although it cannot be used therapeutically, it should serve as a valuable tool for dissecting nuclear export pathways.

Abstract. Expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the presence of the viral trans-activator protein Rev. Rev is localized in the nucleus and binds specifically to the Rev response element (RRE) sequence in viral RNA. Furthermore, the interaction of the Rev activation domain with a cellular cofactor is essential for Rev function in vivo. Using cross-linking experiments and Biospecific Interaction Analysis (BIA) we identify eukaryotic initiation factor 5A (elF-5A) as a cellular factor binding specifically to the HIV-1 Rev activation domain. Indirect immunofluorescence studies demonstrate that a significant fraction of elF-5A localizes to the nucleus. We also provide evidence that Rev transactivation is functionally mediated by elF-5A in Xenopus oocytes. Furthermore, we are able to block Rev function in mammalian cells by antisense inhibition of elF-5A gene expression. Thus, regulation of HIV-1 gene expression by Rev involves the targeting of RREcontaining RNA to components of the cellular translation initiation complex.