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Molecular cloning and cell cycle-dependent expression of mammalian CRM1, a protein involved in nuclear export of proteins - PubMed

  • ️Wed Jan 01 1997

. 1997 Nov 21;272(47):29742-51.

doi: 10.1074/jbc.272.47.29742.

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Molecular cloning and cell cycle-dependent expression of mammalian CRM1, a protein involved in nuclear export of proteins

N Kudo et al. J Biol Chem. 1997.

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Abstract

Crm1 of Schizosaccharomyces pombe, a nuclear protein essential for proliferation and chromosome region maintenance, is a possible target of leptomycin B, an antifungal and antitumor antibiotic with cell cycle-arresting activity. cDNA encoding a human homolog of Crm1 was cloned. Human 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 promoters suppressed cell proliferation in wild-type S. pombe in an expression level-dependent manner. A similar inhibitory effect was also observed for crm1+. Cells overproducing either hCRM1 or S. pombe Crm1 were distinctly larger than uninduced cells and contained compacted and fragmented nuclei. Furthermore, calcofluor staining demonstrated that most of these cells formed two septa per cell and accumulated a large amount of chitin or its related polysaccharides around the septa. Closely similar phenotypes between hCRM1- and S. pombe Crm1-induced cells indicate that the cloned cDNA encodes a functional homolog of S. pombe crm1+. Northern blot analyses with RNAs isolated from synchronized mammalian cells showed that the expression of mammalian CRM1 was initiated in late G1 and reached a peak at G2/M, although its protein level unchanged during the cell cycle. Transient expression of hCRM1 fused to the green fluorescent protein (GFP) in NIH3T3 cells showed that hCRM1 was localized preferentially in the nuclear envelope and was also detectable in the nucleoplasm and the cytoplasm. A crm1 mutation of S. pombe caused nuclear import of a GFP fusion protein containing a nuclear export signal but no change in the distribution of a GFP fusion protein containing a nuclear localization signal. All of these data suggest that CRM1 is a novel cell-cycle regulated gene that is essential for the nuclear export signal-dependent nuclear export of proteins.

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