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 ␣-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.Leukotrienes are lipid messengers that play central roles in immune response and tissue homeostasis (reviewed in Ref. 1). However, abnormal production of leukotrienes contributes to a variety of diseases. The enzyme 5-lipoxygenase (5-LO) 1 catalyzes the initial steps in the synthesis of leukotrienes from arachidonic acid. For this reason, leukotriene synthesis is critically dependent on processes that modulate 5-LO activity.One such process is nuclear import of 5-LO. In leukocytes circulating in peripheral blood, 5-LO is found exclusively in the cytoplasm (2-4). However, import of 5-LO into the nucleus can be induced by recruitment of cells into sites of inflammation (2, 5), by adherence to different surfaces (2, 5, 6), or by cytokine treatment (3). Import of 5-LO is linked to activity since nuclear import of 5-LO can strongly enhance (2, 3) or suppress (5) leukotriene synthesis upon subsequent cell stimulation.The regulation of nuclear import of 5-LO is poorly understood. In particular, our understanding of the regions on 5-LO that act as nuclear import sequences (NISs) is incomplete. Previously, Funk and colleagues (7) identified three candidate regions based on the abundance of the basic amino acids Arg or Lys. Partial mutagenesis of these basic region...

5-Lipoxygenase catalyzes the synthesis of leukotrienes from arachidonic acid. This enzyme can reside either in the cytoplasm or the nucleus; its subcellular distribution is influenced by extracellular factors, and its nuclear import correlates with changes in leukotriene synthetic capacity. To identify sequences responsible for the nuclear import of 5-lipoxygenase, we transfected NIH 3T3 cells and RAW 264.7 macrophages with expression vectors encoding 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 fluorescence, 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 signal. Studies using deletion mutants indicated that this region was necessary for nuclear import of 5-lipoxygenase. Analysis of mutants containing specific amino acid substitutions within this sequence confirmed that it was this sequence that was necessary for nuclear import of 5-lipoxygenase and that a specific arginine residue was critical for this function. As nuclear import of 5-lipoxygenase may regulate leukotriene production, natural or induced mutations in this bipartite nuclear localization sequence may also be important in affecting leukotriene synthesis.Leukotrienes (LTs) 1 are lipid mediators with important roles in immune responsiveness and antimicrobial host defense. However, overproduction of LTs can contribute to a variety of pathophysiological inflammatory processes, including asthma and allergic responses (1). Understanding the regulation of LT synthesis is critical to understanding both normal immune responses and the underlying pathophysiology of a variety of diseases.The synthesis of LTs from arachidonic acid (AA) is initiated by the enzyme 5-lipoxygenase (5-LO). Activation of 5-LO, which is characterized by its translocation to the nuclear envelope (2-5), positions 5-LO close to its substrate and 5-LO activating protein (6 -8) and is essential for LT synthesis. Changes in substrate availability or in the level of expression of proteins essential for AA metabolism (5-LO and/or 5-LO activating protein) have been correlated with the modulation of LT synthetic capacity (9 -14). However, changes in these variables do not account for all observed changes in LT synthetic capacity (15-17).Evidence is accumulating that, even in resting or unstimulated cells, the subcellular distribution of 5-LO is dynamic and may influence LT synthetic capacity. In resting cells, the subcellular distribution of 5-LO is cell type-dependent; 5-LO is predominantly cytoplasmic in peripheral blood neutrophils (18 -20), peripheral blood monocytes (4), and peritoneal macrophages (3), whereas it is found in both the nucleus and cytoplasm of alveolar macrophages (4, 5), mast cells (21), and the mast cell-like rat basophilic leukemia cell line (...

A series of BAL 31 deletions were constructed in the upstream region of the Saccharomyces cerevisiae CYC7 gene to determine sequences required for transcriptional initiation. These deletions identified the TATA box as an alternating A-T sequence at -160 and the initiation sequences as well as the spatial relationship between them. The TATA box was necessary for wild-type levels of expression of the CYC7 gene. Decreasing the distance between the TATA sequence and the initiation site did not alter gene expression, but the site of transcription was shifted 3'-ward. In most cases, transcription initiated at a number of sites, the 5'-most of which was the first suitable site greater than 45 base pairs 3' of the TATA sequence, suggesting a spatial relationship between these sequences, Consensus sequences previously proposed for initiation sites were evaluated with respect to the start sites identified in this study as well as the start sites of other yeast genes.The selection of the initiation site for the transcription of class II genes of higher eucaryotes is governed by an A+T-rich sequence called the TATA box which directs RNA polymerase II to begin transcription 30 base pairs (bp) downstream (2). Deletion of a TATA sequence results in either decreased gene expression, initiation at novel sites, or both (1,9,28,30). If the distance between the TATA sequence and the initiation site is altered, transcription begins at a new location 30 bp downstream from the relocated TATA sequence (1). No sequence preference at the start site is evident, although transcription often begins at an A residue.In Saccharomyces cerevisiae, TATA-like sequences have been identified in the upstream region of most class II genes and are thought to play a role in transcriptional initiation (45). However, for several reasons it is difficult to assess their importance by inspection of the sequence alone. For one, the regions upstream of yeast genes are generally A+T-rich, containing many candidates for TATA boxes. Second, many yeast genes have multiple start sites, making the assignments of TATA boxes to start sites difficult. Finally, in those cases in which the TATA sequence has been studied, there is not a clear relationship between the spacing between the TATA box and the initiation site.Recent studies of the CYC1 (17), HIS3-DEDI (6), HIS4 (35), and PH05 (42) genes have defined a range over which TATA sequences can act; deletions altering the spacing between the TATA sequence and the initiation site point to an effective range of 40 to 120 bp over which TATA sequences can direct transcription. Given the variability in the distance between TATA sequences and the initiation site, it follows that signals contained in sequences at the initiation site must play a role in determining where transcription should begin (4,6,13,17,32,35,42). However, the different studies have identified different sequences. Clearly, more data are required to carry out any comprehensive comparative study.The S. cerevisiae CYC7 gene codes for 5% of the cytochrome c pr...

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Microscopic screening of a collection of cold-sensitive mutants of Saccharomyces cerevisiae led to the identification of a new gene, CDC55, which appears to be involved in the morphogenetic events of the cell cycle. CDC55 maps between CDC43 and CHC1 on the left arm of chromosome VII. At restrictive temperature, the original cdc55 mutant produces abnormally elongated buds and displays a delay or partial block of septation and/or cell separation. A cdc55 deletion mutant displays a cold-sensitive phenotype like that of the original isolate. Sequencing of CDC55 revealed that it encodes a protein of about 60 kDa, as confirmed by Western immunoblots using Cdc55p-specific antibodies. This protein has greater than 50% sequence identity to the B subunits of rabbit skeletal muscle type 2A protein phosphatase; the latter sequences were obtained by analysis of peptides derived from the purified protein, a polymerase chain reaction product, and cDNA clones. An extragenic suppressor of the cdc55 mutation lies in BEM2, a gene previously identified on the basis of an apparent role in bud emergence.