Abstract
Nuclear export signal (NES)-containing proteins are recognized by the NES receptor CRM1/Crm1p (also called exportin 1/Xpo1p). In vertebrates and Schizosaccharomyces pombe, the toxin leptomycin B (LMB) inhibits CRM1-mediated export by interacting directly with CRM1 and disrupting the trimeric Ran-GTP-CRM1-NES export complex. In Saccharomyces cerevisiae, LMB is not toxic and is apparently unable to interact with Crm1p. A second difference between the systems is that LMB has no effect on mRNA export in vertebrate systems, whereas there is evidence that S.cerevisiae Crm1p plays a role in mRNA export. Here we show that a single amino acid change converts S. cerevisiae Crm1p from being LMB insensitive to fully LMB sensitive, indicating that Crm1p is the only relevant LMB target. This new strain has no phenotype, but LMB has a rapid and potent inhibitory effect on NES-mediated export. In situ hybridization assays show that LMB also causes nuclear accumulation of poly(A)+ RNA but with a significant delay compared with the effect on NES-mediated export. Biochemical assays indicate little or no LMB effect on cytoplasmic protein synthesis, indicating that the NES-Crm1p pathway is not a major mRNA export route in S.cerevisiae. We conclude that Crm1p structure and function is conserved from S.cerevisiae to man.
MeSH terms
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Amino Acid Sequence
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Amino Acid Substitution
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Binding Sites
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Biological Transport / drug effects
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Carrier Proteins / chemistry
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Cell Nucleus / metabolism*
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Conserved Sequence / genetics
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Exportin 1 Protein
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Fatty Acids, Unsaturated / pharmacology
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GTP-Binding Proteins / genetics
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GTP-Binding Proteins / physiology
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GTPase-Activating Proteins*
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Genes, Fungal / genetics
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Heat-Shock Proteins / biosynthesis
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Karyopherins*
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Microbial Sensitivity Tests
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Molecular Sequence Data
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Poly A / genetics
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Protein Biosynthesis / genetics
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Protein Sorting Signals / genetics
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Protein Sorting Signals / physiology
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RNA, Messenger / genetics
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RNA, Messenger / metabolism*
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Receptors, Cytoplasmic and Nuclear*
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / growth & development
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins
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Schizosaccharomyces / genetics
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Schizosaccharomyces / metabolism
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Schizosaccharomyces pombe Proteins
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Species Specificity
Substances
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Carrier Proteins
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Fatty Acids, Unsaturated
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GTPase-Activating Proteins
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Heat-Shock Proteins
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Karyopherins
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Protein Sorting Signals
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RNA, Messenger
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RNA1 protein, S cerevisiae
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Receptors, Cytoplasmic and Nuclear
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Recombinant Fusion Proteins
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Saccharomyces cerevisiae Proteins
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Schizosaccharomyces pombe Proteins
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rna1 protein, S pombe
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Poly A
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GTP-Binding Proteins
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leptomycin B