Analysis of mechanism of tress-responsive and temperature-sensitive nuclear transport

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The target molecule of leptomycin B (LMB), a potent antitumor agent, was genetically and biochemically identified as CRM1 , a protein reported as being required for chromosome structure control. We showed that CRM1 was a receptor for the nuclear export signal (NES) and that LMB inhibited nuclear export of proteins. It therefore became able to easily determine whether a protein of interest can be exported from the nucleus by using LMB. Mouse temperature-sensitive p53^<Val135> (tsp53) accumulates in the nucleus and acts as a wild-type at 32℃, while it is sequestered in the cytoplasm at 37℃. The cytoplasmic tsp53 relocalized into the nucleus upon inhibition of the nudear export by LMB at 37℃, whereas a mutation in a major bipartite NLS caused constitutive cytoplasmic localization, indicating that it shuttled between the cytoplasm and the nucleus by its NES and NLS rather than tethered to cytoplasmic structures. We showed that the association with the Hsc70-containing complex prevents the NLS from the access of the import receptor through the C- terminal region of tsp53 at 37℃, whereas its dissociation at 32℃ allows rapid nuclear import. Using LMB, we identified a novel NES in the fission yeast transcription factor Pap1 , the function of which is abolished by oxidative stress in a manner conserved in eukaryotes. A single mutation in Crm1 (Cys-529 to Ser) caused a marked decrease in exporting Pap1 but not other typical NESs. These results suggest that the cysteine residue in Crm1 is involved in the stress-responsive nuclear export of Pap1.