Crm1-mediated nuclear export of the Schizosaccharomyces pombe transcription factor Cuf1 during a shift from low to high copper concentrations.
In this study, we examine the fate of the nuclear pool of the Schizosaccharomyces pombe transcription factor Cuf1 in response to variations in copper levels. A nuclear pool of Cuf1-green fluorescent protein (GFP) was generated by expressing a functional cuf1(+)-GFP allele in the presence of a copper chelator. We then extinguished cuf1(+)-GFP expression and tracked the changes in the localization of the nuclear pool of Cuf1-GFP in the presence of low or high copper concentrations. Treating cells with copper as well as silver ions resulted in the nuclear export of Cuf1. We identified a leucine-rich nuclear export signal (NES), (349)LAALNHISAL(358), within the C-terminal region of Cuf1. Mutations in this sequence abrogated Cuf1 export from the nucleus. Furthermore, amino acid substitutions that impair Cuf1 NES function resulted in increased target gene expression and a concomitant cellular hypersensitivity to copper. Export of the wild-type Cuf1 protein was inhibited by leptomycin B (LMB), a specific inhibitor of the nuclear export protein Crm1. We further show that cells expressing a temperature-sensitive mutation in crm1(+) exhibit increased nuclear accumulation of Cuf1 at the nonpermissive temperature. Although wild-type Cuf1 is localized in the nucleus in both conditions, we observed that the protein can still be inactivated by copper, resulting in the repression of ctr4(+) gene expression in the presence of exogenous copper. These results demonstrate that nuclear accumulation of Cuf1 per se is not sufficient to cause the unregulated expression of the copper transport genes like ctr4(+). In addition to nuclear localization, a functional Cys-rich domain or NES element in Cuf1 is required to appropriately regulate copper transport gene expression in response to changes in intracellular copper concentration.
Beaudoin
Jude
J
Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, 3001 12e Ave Nord, Sherbrooke, Québec J1H 5N4, Canada.
Labbé
Simon
S
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000595:Amino Acid Sequence; D000596:Amino Acids; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D003300:Copper / Q000494:pharmacology*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D028884:Karyopherins / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D049790:Nuclear Export Signals; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000378:metabolism*; D012834:Silver / Q000494:pharmacology; D013696:Temperature; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D000097606:Exportin 1 Protein
D000596:Amino Acids; D003300:Copper; D005231:Fatty Acids, Unsaturated; D049452:Green Fluorescent Proteins; D028884:Karyopherins; D049790:Nuclear Export Signals; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D012834:Silver; D014157:Transcription Factors; D000097606:Exportin 1 Protein; C474030:Cuf1 protein, S pombe; C038753:leptomycin B