The membrane organization of leukotriene synthesis

Abstract

Cell signaling leading to the formation of leukotriene (LT)C(4) requires the localization of the four key biosynthetic enzymes on the outer nuclear membrane and endoplasmic reticulum. Whether any macromolecular organization of these proteins exists is unknown. By using fluorescence lifetime imaging microscopy and biochemical analysis, we demonstrate the presence of two distinct multimeric complexes that regulate the formation of LTs in RBL-2H3 cells. One complex consists of multimers of LTC(4) synthase and the 5-lipoxygenase activating protein (FLAP). The second complex consists of multimers of FLAP. Surprisingly, all LTC(4) synthase was found to be in association with FLAP. The results indicate that the formation of LTC(4) and LTB(4) may be determined by the compartmentalization of biosynthetic enzymes in discrete molecular complexes.

Fig. 1.

FLIM analysis of the interactions of FLAP and LTC₄ synthase. (A) Pseudocolor imaging of the interaction of LTC₄ synthase and FLAP analyzed by live-cell FLIM. COS cells were transfected with 4 μg of individual plasmids in six-well culture dishes and were analyzed 24 h later. Transfected or cotransfected COS cells were analyzed by time-domain FLIM in PBS by using double-exponential decay algorithms. The data are from a representative experiment. Pseudocolor imaging represents the weighted average lifetime for two lifetime values per pixel. The lifetime pseudocolor scale on the bottom depicts the fluorescence lifetime over a scale of 2,500 ps (blue) to 250 ps (orange), which depict increasingly strong interactions. (AA) FLIM of ECFP-FLAP and EYFP-FLAP. (AB) FLIM of ECFP-FLAP alone. (AC) FLIM of ECFP-LTC₄ synthase and EYFP-LTC₄ synthase. (AD) FLIM of ECFP-LTC₄ synthase alone. (AE) FLIM of ECFP-FLAP and EYFP-LTC₄ synthase. (AF) FLIM of soluble ECFP and EYFP proteins coexpressed in a single cell. (Scale bars, 10 μm in AB; 40 μm in AC.) (B) Bar graph analysis of FLIM data. The data represent the mean ± SD for 5-17 separate cells taken from four to six experiments, with the exception of the ECFP-EYFP fusion protein where the data represent the mean of two cells from two experiments. t₁, the fast-lifetime component of the average lifetime; LTC₄S, LTC₄ synthase.

Fig. 2.

FLAP and LTC₄ synthase interact. (A) Extracts of RBL-2H3 cells were immunoprecipitated (IP) with antibody to LTC₄ synthase (L) or control nonimmune IgG (C). Immunoprecipitates were resolved by using SDS/12% gels, and were then analyzed by Western blotting with antibodies to LTC₄ synthase or FLAP (F). (B) Avi-tag/FLAG-FLAP and His₆-tagged LTC₄ synthase were immunoprecipitated from extracts of Sf9 cell microsomes by using antibodies to FLAP, LTC₄ synthase, FLAG (FL), or the His₆ epitope (H). The immunoprecipitates were resolved on two identical gels, which were analyzed by Western blotting using antibodies to LTC₄ synthase or FLAP (Right). Microsomes (M) and 100 ng of pure protein (F^* and L^*) were also probed as controls. (C) Two populations of FLAP exist. Extracts of RBL-2H3 cells were immunoprecipitated with control IgG, antibody to LTC₄ synthase, or antibody to FLAP. The resulting supernatants and pellets were resolved by SDS/12% gels and were then analyzed by Western blotting using the antibodies shown at Right. (D) Northern blot analysis of mRNA for LTC₄ synthase, FLAP, 5-LO, and a representative GAPDH control. mRNA sizes (kb) are shown at Right. A representative lane of GAPDH analysis is shown; all lanes were indistinguishable. (E) Anti-LTC₄ synthase and anti-FLAP antibodies have equal titers. Identical concentrations of homogeneous purified His-epitope-tagged LTC₄ synthase or avitag-FLAP were separated by SDS/12% gels, were electroblotted, and were probed with antisera to LTC₄ synthase or FLAP at dilutions of 1:100 or 1:200, respectively. Immunoprecipitates using antisera against FLAP, LTC₄ synthase, FLAG, or His₆ were analyzed with antibodies to FLAP or LTC₄ synthase.

Fig. 3.

Endogenous FLAP exists as multimers. RBL cells were analyzed directly (lane 1) or after crosslinking in situ (lanes 2 and 3) with the reducible, membrane permeant crosslinker dimethyl-3-3′-dithiobispropionimidate·2 HCl. Cell extracts were analyzed directly (lane 3) or were resolved after reduction with 2-mercaptoethanol by using SDS/12% gels (lane 2). They were then analyzed by Western blotting with antibody to FLAP. M, monomers; D, dimers; T, trimers.

Fig. 4.

The potential membrane interactions of FLAP and LTC₄ synthase. Because FLAP (F, dark blue) is in excess to LTC₄ synthase (L, light blue), there is no free LTC₄ synthase. Two populations of FLAP are predicted to exist within membranes, one that is dedicated to the formation of LTC₄, and one that is dedicated to the formation of LTB₄. The possibility of both dimers and trimers contributing to the formation of leukotrienes is shown.?, the unknown composition of trimers.