Identification of a bipartite nuclear localization sequence necessary for nuclear import of 5-lipoxygenase

5-Lipoxygenase catalyzes the synthesis of leukotrienes from arachidonic aci d. This enzyme can reside either in the cytoplasm or the nucleus; its subce llular distribution is influenced by extracellular factors, and its nuclear import correlates with changes in leukotriene synthetic capacity. To ident ify sequences responsible for the nuclear import of B-lipoxygenase, we tran sfected NIH 3T3 cells and RAW 264.7 macrophages with expression vectors enc oding various 5-lipoxygenase constructs fused to green fluorescent protein. Overexpression of wild type 5-lipoxygenase with or without fusion to green fluorescent protein resulted in a predominantly intranuclear pattern of fl uorescence, similar to the distribution of native 5-lipoxygenase in primary alveolar macrophages. Within the 5-lipoxygenase protein is a sequence (Arg (638)-Lys(655)) that closely resembles a bipartite nuclear localization sig nal. Studies using deletion mutants indicated that this region was necessar y for nuclear import of 5-lipoxygenase. Analysis of mutants containing spec ific amino acid substitutions within this sequence confirmed that it was th is sequence that was necessary for nuclear import of 5-Lipoxygenase and tha t a specific arginine residue was critical for this function. As nuclear im port of 5-lipoxygenase may regulate leukotriene production, natural or indu ced mutations in this bipartite nuclear localization sequence may also be i mportant in affecting leukotriene synthesis.