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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Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's α kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its α kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila MEI-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its α kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. Our results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin.

Appropriate subcellular localization is crucial for regulation of NF-B function. Herein, we show that latent NF-B complexes can enter and exit the nucleus in preinduction states. The nuclear export inhibitor leptomycin B (LMB) sequestered NF-B͞IB␣ complexes in the nucleus. Using deletion and site-directed mutagenesis, we identified a previously uncharacterized nuclear export sequence in residues 45-54 of IB␣ that was required for cytoplasmic localization of inactive complexes. This nuclear export sequence also caused nuclear exclusion of heterologous proteins in a LMB-sensitive manner. Importantly, a LMB-insensitive CRM1 mutant (Crm1-K1) abolished LMB-induced nuclear accumulation of the inactive complexes. Moreover, a cell-permeable p50 NF-B nuclear localization signal peptide also blocked these LMB effects. These results suggest that NF-B͞IB␣ complexes shuttle between the cytoplasm and nucleus by a nuclear localization signal-dependent nuclear import and a CRM1-dependent nuclear export. The LMB-induced nuclear complexes could not bind DNA and were inaccessible to signaling events, because LMB inhibited NF-B activation without affecting the subcellular localization of upstream kinases IKK␤ and NIK. Our findings indicate that the dominant nuclear export over nuclear import contributes to the largely cytoplasmic localization of the inactive complexes to achieve efficient NF-B activation by extracellular signals. R egulatory pathways can be modulated by the subcellular compartmentalization of individual components. A prototypic example is signal-induced activation of the transcription factor NF-B that plays an important role in immune and inflammatory responses and the regulation of apoptosis (1, 2). In unstimulated cells, inactive NF-B preexists in the cytoplasm associated with its inhibitor IB (3). On exposure to extracellular signals, a series of biochemical events targets the inhibitor protein for degradation, allowing the NF-B to migrate into the nucleus to regulate gene expression.Cis-acting elements of IB govern its protein stability and subcellular localization (1, 2). IB␣, the most studied IB family member, is composed minimally of three domains: an Nterminal regulatory domain that controls signal-dependent degradation, a central ankyrin repeat domain (ARD) that is necessary for NF-B binding, and a C-terminal region rich in proline, glutamate/aspartate, serine, and threonine regulating basal turnover. An additional sequence, a leucine-rich nuclear export sequence (NES) within the last ankyrin repeat, is postulated to function during the termination of NF-B activity (4). Activated NF-B stimulates the synthesis of IB␣ mRNA (5, 6), and newly synthesized IB␣ proteins can enter the nucleus to bind to and remove NF-B from gene promoters (7). It is believed that the C-terminal NES (C-NES) of IB␣ can actively export these IB␣͞NF-B complexes out to the cytoplasm to restore the preinduction state of the complexes, a process known as postinduction repression (4).The leucine-rich NES is a highly conserved sequence use...