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

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

^*Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan; and ^†Novartis Forschungsinstitut, Brunner Strasse 59, A-1235 Vienna, Austria

Edited by Günter Blobel, The Rockefeller University, New York, NY, and approved June 10, 1999 (received for review April 12, 1999)

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. ¹H 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 α,β-unsaturated δ-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.

Footnotes

↵ ‡ To whom reprint requests should be addressed. E-mail: ayoshida{at}hongo.ecc.u-tokyo.ac.jp.
This paper was submitted directly (Track II) to the Proceedings Office.
Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. AB027496 (crm1-809), AB027497 (crm1-F1), and AB027498 (crm1-K1)].
ABBREVIATIONS:

CCR,

central conserved region;

CRM1,

chromosomal region maintenance 1;

CRIME,

CRM1, importin-β, etc;

GFP,

green fluorescent protein;

GSH,

glutathione;

LMB,

leptomycin B;

NEM,

N-ethylmaleimide;

NES,

nuclear export signal;

NLS,

nuclear localization signal;

GST,

glutathione S-transferase