Structural and functional criteria reveal a new nuclear import sequence on the 5-lipoxygenase protein.
Leukotrienes are lipid mediators with important roles in immunity. The enzyme 5-lipoxygenase initiates leukotriene synthesis; nuclear import of 5-lipoxygenase modulates leukotriene synthetic capacity. In this study, we used structural and functional criteria to identify potential nuclear import sequences. Specifically, we sought basic residues that 1) were common to different 5-lipoxygenases but not shared with other lipoxygenases, 2) were found on random coil/loop structures, and 3) could be replaced without eliminating catalytic activity. Application of these criteria to the putative bipartite nuclear import sequence of 5-lipoxygenase revealed that this region formed an alpha-helix rather than a random coil, that the critical residue arginine 651 serves a structural role, and that mutation of this residue eliminated catalytic activity. A previously unrecognized region corresponding to residues 518-530 on human 5-lipoxygenase was found to be unique to 5-lipoxygenase and on a random coil. This region alone was sufficient to drive import of green fluorescent protein to the same degree as complete 5-lipoxygenase. Replacement of basic residues in this region of the complete protein was capable of eliminating nuclear import without abolishing catalytic activity. Surprisingly, two subpopulations of cells expressing 5-lipoxygenase with this mutated region could be discerned: those with strongly impaired import and those with normal import. Taken together, these results show that the previously identified region with a bipartite motif is not a functional import sequence, whereas the newly identified basic region constitutes a true nuclear import sequence. Moreover, we suggest that another sequence that can mediate nuclear import of 5-lipoxygenase remains to be identified.
Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Health System, Ann Arbor, Michigan 48109-0642, USA.
Sandra M
SM
Jones
Ming
M
Luo
Annette M
AM
Healy
Marc
M
Peters-Golden
Thomas G
TG
Brock
D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry* / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D015289:Leukotrienes / Q000096:biosynthesis; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D021382:Protein Sorting Signals; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment
D015289:Leukotrienes; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001094:Arachidonate 5-Lipoxygenase