Nuclear-localization-signal-dependent and nuclear-export-signal-dependent mechanisms determine the localization of 5-lipoxygenase.
5-Lipoxygenase (5-LO) metabolizes arachidonic acid to leukotriene A4, a key intermediate in leukotriene biosynthesis. To explore the molecular mechanisms of its cell-specific localization, a fusion protein between green fluorescent protein (GFP) and human 5-LO (GFP-5LO) was expressed in various cells. GFP-5LO was localized in the cytosol in HL-60 cells and in both the nucleus and the cytosol in RBL (rat basophilic leukaemia) cells, similarly to the native enzyme in these cells. The localization of GFP fusion proteins for mutant 5-LOs in a putative bipartite nuclear localization signal (NLS), amino acids 638-655, in Chinese hamster ovary (CHO)-K1 and Swiss3T3 cells revealed that this motif is important for the nuclear localization of 5-LO. A GFP fusion protein of this short peptide localized consistently in the nucleus. Leptomycin B, a specific inhibitor of nuclear export signal (NES)-dependent transport, diminished the cytosolic localization of 5-LO in HL-60 cells and that of GFP-5LO in CHO-K1 cells, suggesting that an NES-system might also function in determining 5-LO localization. Analysis of the localization of 5-LO during the cell cycle points to a controlled movement of this enzyme. Thus we conclude that a balance of NLS- and NES-dependent mechanisms determines the cell-type-specific localization of 5-LO, suggesting a nuclear function for this enzyme.
Hanaka
Hiromi
H
Department of Biochemistry, Gunma University School of Medicine, 3-39-22 Showamachi, Maebashi, Gunma 371-8511, Japan.
Shimizu
Takao
T
Izumi
Takashi
T
D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000096:biosynthesis* / Q000737:chemistry; D016466:CHO Cells; D002453:Cell Cycle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D018922:HL-60 Cells; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019913:Nuclear Localization Signals / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D005231:Fatty Acids, Unsaturated; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001094:Arachidonate 5-Lipoxygenase; C038753:leptomycin B