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 inhibit the function of CRM1, a receptor for the nuclear export signal (NES). Here we show evidence that LMB binds directly to CRM1 and that CRM1 is essential for NES-dependent nuclear export of proteins in both yeast and mammalian cells. Binding experiments with a biotinylated derivative of LMB and a HeLa cell extract led to identifying CRM1 as a major protein that bound to the LMB derivative. Microinjection of a purified anti-human CRM1 antibody into the mammalian nucleus specifically inhibited nuclear export of NES-containing proteins, as did LMB. Consistent 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 LMB was also observed in fission yeast. The fission yeast crm1 mutant was defective in the nuclear export of NES-fused proteins, but not in the import of nuclear localization signal (NLS)-fused proteins. Interestingly, a protein containing both NES and NLS, which is expected 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 LMB and is an essential factor for nuclear export of proteins in eukaryotes.
Kudo
N
N
Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113, Japan.
Wolff
B
B
Sekimoto
T
T
Schreiner
E P
EP
Yoneda
Y
Y
Yanagida
M
M
Horinouchi
S
S
Yoshida
M
M
D001693:Biological Transport, Active / Q000187:drug effects; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002467:Cell Nucleus / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D015694:Gene Products, rev / Q000235:genetics / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins; D019913:Nuclear Localization Signals / Q000187:drug effects / Q000502:physiology*; D009687:Nuclear Proteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins / Q000235:genetics; D012568:Schizosaccharomyces / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D000097606:Exportin 1 Protein
D002352:Carrier Proteins; D005231:Fatty Acids, Unsaturated; D015694:Gene Products, rev; D028884:Karyopherins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D000097606:Exportin 1 Protein; C038753:leptomycin B