MOLECULAR-CLONING AND CELL CYCLE-DEPENDENT EXPRESSION OF MAMMALIAN CRM1, A PROTEIN INVOLVED IN NUCLEAR EXPORT OF PROTEINS

Crm1 of Schizosaccharomyces pombe, a nuclear protein essential for pro liferation and chromosome region maintenance, is a possible target of leptomycin B, an antifungal and antitumor antibiotic with cell cycle-a rresting activity, cDNA encoding a human homolog of Crm1 was cloned, H uman CRM1 (hCRM1) consisted of 1071 amino acids, of which the sequence showed 52% homology with S. pombe Crm1. hCRM1 weakly complemented the cold sensitive mutation of S. pombe crm1-809, as did S. pombe crm1(+) , Overproduction of hCRM1 under the control of a series of nmt1 promot ers suppressed cell proliferation in wild-type S. pombe in an expressi on level-dependent manner, A similar inhibitory effect was also observ ed for crm1(+). Cells overproducing either hCRM1 or S. pombe Crm1 were distinctly larger than uninduced cells and contained compacted and fr agmented nuclei, Furthermore, calcofluor stain ing demonstrated that m ost of these cells formed two septa per cell and accumulated a large a mount of chitin or its related polysaccharides around the septa, Close ly similar phenotypes between hCRM1- and S. pombe Crm1-induced cells i ndicate that the cloned cDNA encodes a functional homolog of S. pombe crm1(+). Northern blot analyses with RNAs isolated from synchronized m ammalian cells showed that the expression of mammalian CRM1 was initia ted in late G(1) and reached a peak at G(2)/M, although its protein le vel unchanged during the cell cycle, Transient expression of hCRM1 fus ed to the green fluorescent protein (GFP) in NIH3T3 cells showed that hCRM1 was localized preferentially in the nuclear envelope and was als o detectable in the nucleoplasm and the cytoplasm, A crm1 mutation of S. pombe caused nuclear import of a GFP fusion protein containing a nu clear export signal but no change in the distribution of a GFP fusion protein containing a nuclear localization signal, All of these data su ggest that CRM1 is a novel cell-cycle regulated gene that is essential for the nuclear export signal-dependent nuclear export of proteins.