Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region

Abstract

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 ...

Firefly luciferase mutants as sensors of proteome stress

“…After exit from the nucleus, the hydrolysis of RanGTP to RanGDP by RanGTPase causes the dissociation of CRM1-cargo complex and thereby releases the cargo into the cytoplasm. This export cycle can be inhibited by the small molecule leptomycin B (LMB) which alkylates Cys529 of CRM1 and blocks its function (Kudo et al, 1999;Kudo et al, 1998).…”

Involvement of protein phosphatase 2A nuclear accumulation and subsequent inactivation of activator protein-1 in leptomycin B-inhibited cyclin D1 expression

“…LMB was shown to interact directly with CRM1 and disrupt the interaction between CRM1 and NES, resulting in inhibition of the effects of CRM1 (Nishi et al, 1994;Fornerod et al, 1997;Kudo et al, 1998Kudo et al, , 1999. Since its discovery, LMB has become an important 'tool compound' for studying the regulation of nucleocytoplasmic shuttling proteins.…”

Nuclear export inhibitor leptomycin B induces the appearance of novel forms of human Mdm2 protein

“…LMB is thought to function primarily as an inhibitor of the export of proteins from the nucleus to the cytoplasm because of its ability to interact with and impair the function of the nuclear export factor crm-1 (Kudo et al, 1999). Although other effects of LMB that could lead to a DNA damage response cannot be excluded, LMB is likely to be a very potent inducer of the p53 response, which does not act directly through the DNA damage pathway (Lain et al, 1999b).…”