LEPTOMYCIN-B INHIBITION OF SIGNAL-MEDIATED NUCLEAR EXPORT BY DIRECT BINDING TO CRM1

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 inhibi t the function of CRM1, a receptor for the nuclear export signal (NES) . Here we show evidence that LIMB binds directly to CRMI1 and that CRM 1 is essential for NES-dependent nuclear export of proteins in both ye ast and mammalian cells. Binding experiments with a biotinylated deriv ative of LMB and a HeLa cell extract led to identifying CRM1 as a majo r protein that bound to the LMB derivative. Microinjection of a purifi ed anti-human CRM1 antibody into the mammalian nucleus specifically in hibited nuclear export of NES-containing proteins, as did LMB. Consist ent 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 LIMB was also observed in fission yeast. The fission yeast cr m1 mutant was defective in the nuclear export of NES-fused proteins, b ut not in the import of nuclear localization signal (NLS)-fused protei ns. Interestingly, a protein containing both NES and NLS, which is exp ected 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 LIMB and is an essential factor for nuclear export of proteins in eukaryotes. (C) 1998 Academic Press.