Sequence of Crm1/exportin 1 mutant alleles reveals critical sites associated with multidrug resistance

We have previously shown that genes involved in a novel pathway of multidru g resistance (MDR) in the fission yeast Schizosaccharomyces pombe are funct ionally conserved in human cells (V. Spataro et al. (1997) J Biol Chem 272: 30470-30475). The human homologue of one of these genes, hCRM1, has recent ly been identified and found to function in nucleocytoplasmic export, a pro cess which controls the subcellular localization and hence activity of a nu mber of key cell cycle regulators and transcription factors. Several mutant alleles of crm1 confer a phenotype of MDR in S. pombe, through the nuclear accumulation of the AP-1 transcription factor Pap1. We therefore sequenced mutations of crm1 in fission yeast in order to guide the search for analog ous hCRM1 mutations which could play a role in tumour-drug resistance. Fift een yeast crm1 mutants were assessed by PCR and DNA sequencing. Four mis-se nse mutations were identified in the open reading frame, three of which (G to A transitions at nucleotide positions 385, 895 and 1,288) were capable o f conferring the MDR phenotype alone. For three of the four mutations found , the corresponding amino acid changes affect residues which are conserved in the human homologue hCRM1 and lie in highly conserved regions of the CRM 1 protein. We analysed the corresponding hCRM1 coding regions by RT-PCR and sequencing in a panel of ten tumour cell lines, including three ovarian li nes resistant either to cisplatin or paclitaxel, or to both and one MDR bre ast cancer cell line with nuclear accumulation of the transcription factor YB-1. No hCRM1 mutations were found in the three cDNA fragments examined in this panel of tumour cell lines. However, the identification of amino acid residues within the CRM1 protein that are critical for the export of the M DR-associated transcription factor Pap1 in fission yeast can guide further analysis of hCRM1 mutations in tumours with a MDR phenotype.