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Identification of regions of leukotriene C 4 synthase which direct the enzyme to its nuclear envelope localization
Author(s) -
Svartz Jesper,
Hallin Elisabeth,
Shi Yixuan,
Söderström Mats,
Hammarström Sven
Publication year - 2006
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.20880
Subject(s) - amino acid , endoplasmic reticulum , transmembrane protein , biochemistry , chemistry , transmembrane domain , arachidonic acid , peptide sequence , hek 293 cells , microbiology and biotechnology , enzyme , biology , gene , receptor
Leukotrienes (LTs) are fatty acid derivatives formed by oxygenation of arachidonic acid via the 5‐lipoxygenase (5‐LO) pathway. Upon activation of inflammatory cells 5‐LO is translocated to the nuclear envelope (NE) where it converts arachidonic acid to the unstable epoxide LTA 4 . LTA 4 is further converted to LTC 4 by conjugation with glutathione, a reaction catalyzed by the integral membrane protein LTC 4 synthase (LTC4S), which is localized on the NE and endoplasmic reticulum (ER). We now report the mapping of regions of LTC4S that are important for its subcellular localization. Multiple constructs encoding fusion proteins of green fluorescent protein (GFP) as the N‐terminal part and various truncated variants of human LTC4S as C‐terminal part were prepared and transfected into HEK 293/T or COS‐7 cells. Constructs encoding hydrophobic region 1 of LTC4S (amino acids 6–27) did not give distinct membrane localized fluorescence. In contrast hydrophobic region 2 (amino acids 60–89) gave a localization pattern similar to that of full length LTC4S. Hydrophobic region 3 (amino acids 114–135) directed GFP to a localization indistinguishable from that of full length LTC4S. A minimal directing sequence, amino acids 117–132, was identified by further truncation. The involvement of the hydrophobic regions in the homo‐oligomerization of LTC4S was investigated using bioluminescence resonance energy transfer (BRET) analysis in living cells. BRET data showed that hydrophobic regions 1 and 3 each allowed oligomerization to occur. These regions most likely form transmembrane helices, suggesting that homo‐oligomerization of LTC4S is due to helix–helix interactions in the membrane. J. Cell. Biochem. 98: 1517–1527, 2006. © 2006 Wiley‐Liss, Inc.