SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2028881470> ?p ?o ?g. }

Showing items 1 to 100 of ±115 with 100 items per page.

W2028881470 endingPage "28908" @default.
W2028881470 startingPage "28902" @default.
W2028881470 abstract "Leukotriene C4(LTC4) synthase conjugates LTA4 with GSH to form LTC4. Determining the site of LTC4synthesis and the topology of LTC4 synthase may uncover unappreciated intracellular roles for LTC4, as well as how LTC4 is transferred to its export carrier, the multidrug resistance protein-1. We have determined the membrane localization of LTC4 synthase by immunoelectron microscopy. In contrast to the closely related five-lipoxygenase-activating protein, LTC4 synthase is distributed in the outer nuclear membrane and peripheral endoplasmic reticulum but is excluded from the inner nuclear membrane. We have combined immunofluorescence with differential membrane permeabilization to determine the topology of LTC4 synthase. The active site of LTC4 synthase is localized in the lumen of the nuclear envelope and endoplasmic reticulum. These results indicate that the synthesis of LTB4 and LTC4 occurs in different subcellular locations and suggests that LTC4 must be returned to the cytoplasmic side of the membrane for export by multidrug resistance protein-1. The differential localization of two very similar integral membrane proteins suggests that mechanisms other than size-dependent exclusion regulate their passage to the inner nuclear membrane. Leukotriene C4(LTC4) synthase conjugates LTA4 with GSH to form LTC4. Determining the site of LTC4synthesis and the topology of LTC4 synthase may uncover unappreciated intracellular roles for LTC4, as well as how LTC4 is transferred to its export carrier, the multidrug resistance protein-1. We have determined the membrane localization of LTC4 synthase by immunoelectron microscopy. In contrast to the closely related five-lipoxygenase-activating protein, LTC4 synthase is distributed in the outer nuclear membrane and peripheral endoplasmic reticulum but is excluded from the inner nuclear membrane. We have combined immunofluorescence with differential membrane permeabilization to determine the topology of LTC4 synthase. The active site of LTC4 synthase is localized in the lumen of the nuclear envelope and endoplasmic reticulum. These results indicate that the synthesis of LTB4 and LTC4 occurs in different subcellular locations and suggests that LTC4 must be returned to the cytoplasmic side of the membrane for export by multidrug resistance protein-1. The differential localization of two very similar integral membrane proteins suggests that mechanisms other than size-dependent exclusion regulate their passage to the inner nuclear membrane. leukotriene arachidonic acid lipoxygenase five-lipoxygenase-activating protein cytochrome P450 streptolysin O multidrug resistance protein Chinese hamster ovary phosphate-buffered saline 1,4-piperazinediethanesulfonic acid electron microscopy enhanced green fluorescent protein HEPES-buffered Ca2+- and Mg2+-free Hanks' solution Leukotrienes (LTs)1 are proinflammatory products of arachidonic acid (AA) metabolism. LTC4, the parent sulfidopeptide LT, is converted to LTD4 by γ-glutamyl transpeptidase. LTC4 and LTD4 have been implicated in the pathogenesis of asthma, renal disease (1Lewis R.A. Austen K.F. Soberman R.J. N. Engl. J. Med. 1990; 323: 645-655Crossref PubMed Scopus (1166) Google Scholar), and dendritic cell trafficking (2Robbiani D.F. Finch R.A. Jager D. Muller W.A. Sartorelli A.C. Randolph G.J. Cell. 2000; 103: 757-768Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). LTC4is formed in four biochemical steps. First, cytoplasmic phospholipase A2 releases AA from phospholipids (3Clark J.D. Lin L.L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 6: 1043-1051Abstract Full Text PDF Scopus (1454) Google Scholar, 4Qiu Z.H. Gijon M.A. de Carvalho M.S. Spencer D.M. Leslie C.C. J. Biol. Chem. 1998; 273: 8203-8211Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Five-lipoxygenase-activating protein (FLAP) then mediates the interaction between five-lipoxygenase (5-LO) and AA (5Dixon R.A. Diehl R.E. Opas E. Rands E. Vickers P.J. Evans J.F. Gillard J.W. Miller D.K. Nature. 1990; 343: 282-284Crossref PubMed Scopus (647) Google Scholar, 6Miller D.K. Gillard J.W. Vickers P.J. Sadowski S. Leveille C. Mancini J.A. Charleson P. Dixon R.A. Ford-Hutchinson A.W. Fortin R. Nature. 1990; 343: 278-281Crossref PubMed Scopus (379) Google Scholar, 7Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heibein J.A. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Crossref PubMed Scopus (358) Google Scholar, 8Mancini J.A. Abromowitz M. Cox M.E. Wong E. Charleson S. Perrier H. Wang Z. Peptiboon P. Vickers P. FEBS Lett. 1993; 318: 277-281Crossref PubMed Scopus (180) Google Scholar) yielding both 5(S)-hydroperoxyeicosatetraenoic acid and LTA4 (9Rouzer C.A. Matsumoto T. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 857-861Crossref PubMed Scopus (268) Google Scholar). LTC4 synthase then catalyzes the conjugation of GSH with LTA4 to form LTC4 (10Penrose J.F. Gagnon L. Goppelt-Struebe M. Myers P. Lam B.K. Jack R.M. Austen K.F. Soberman R.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11603-11606Crossref PubMed Scopus (67) Google Scholar,11Lam B.K. Penrose J.F. Freeman G.F. Austen K.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7663-7667Crossref PubMed Scopus (247) Google Scholar). LTC4 is exported from cells by the plasma membrane protein MRP-1 (12Lam B.K. Owen Jr., W.F. Austen K.F. Soberman R.J. J. Biol. Chem. 1989; 264: 12885-12889Abstract Full Text PDF PubMed Google Scholar, 13Jedlitschky G. Buchholz U. Keppler D. Eur. J. Biochem. 1994; 220: 599-606Crossref PubMed Scopus (148) Google Scholar, 14Leir I. Jedlitschky G. Bucholz U. Cole S.P. Deeley R.G. Keppler D. J. Biol. Chem. 1994; 269: 27807-27810Abstract Full Text PDF PubMed Google Scholar), and cells from MRP-1 knockout mice do not export LTC4 (15Wijnholds J. Evers R. van Leusden M.R. Mol C.A. Zaman G.J. Mayer U. Beijnen J.H. van der Valk M. P. Krimpenfort P. Borst P. Nat. Med. 1997; 3: 1275-1279Crossref PubMed Scopus (399) Google Scholar).A major strategy used by cells to prevent the formation of these bioactive molecules under resting conditions is compartmentalization of the biosynthetic enzymes. In circulating, quiescent peripheral blood cells, both cytoplasmic phospholipase A2 and 5-LO are localized to the cytoplasmic compartment, whereas FLAP is localized to the inner and outer nuclear membrane, preventing the release of AA and the formation of LTs. When leukocytes and mast cells are activated, cytoplasmic phospholipase A2 translocates to the nuclear envelope. At the same time, 5-LO translocates to the inner and outer nuclear membranes. The association of 5-LO with these membranes is regulated, in part, by FLAP (7Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heibein J.A. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Crossref PubMed Scopus (358) Google Scholar, 16Glover S. Bayburt T. Jonas M. Chi E. Chi E. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15567Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). LTC4 synthase and FLAP are closely related 17-kDa transmembrane proteins, and both are predicted to have three hydrophobic domains. The region that extends from the first predicted hydrophilic loop to the third hydrophobic domain is highly homologous between the two proteins (Fig.1). The localization of LTC4 synthase has not been characterized in detail. FLAP is distributed between the inner and outer nuclear membranes and peripheral ER. The first hydrophilic loop of FLAP was localized to the lumen of the nuclear membrane (7Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heibein J.A. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Crossref PubMed Scopus (358) Google Scholar), but the topology of the second hydrophilic loop and the C and N termini of FLAP are unknown, although a model with three transmembrane domains was proposed (8Mancini J.A. Abromowitz M. Cox M.E. Wong E. Charleson S. Perrier H. Wang Z. Peptiboon P. Vickers P. FEBS Lett. 1993; 318: 277-281Crossref PubMed Scopus (180) Google Scholar) (Fig. 1 A). The first hydrophilic loop of LTC4 synthase binds LTA4 (8Mancini J.A. Abromowitz M. Cox M.E. Wong E. Charleson S. Perrier H. Wang Z. Peptiboon P. Vickers P. FEBS Lett. 1993; 318: 277-281Crossref PubMed Scopus (180) Google Scholar, 17Lam B.K. Penrose J.F. Xu K. Baldasaro M.H. Austen K.F. J. Biol. Chem. 1997; 272: 13923-13928Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). GSH conjugation is determined by amino acid residue Tyr93 in the second hydrophilic loop, suggesting that the hydrophilic loops are on the same, cytoplasmic side of the membrane (Fig. 1 B). No experimental determination of the topology has been made to support this model, and other relationships between the hydrophilic loops and the C-and N termini may exist (Fig.1, C–G).We have analyzed the distribution and topology of LTC4synthase using a combination of immunofluorescence, confocal, and electron microscopy combined with differential membrane permeabilization by streptolysin O (SLO). Surprisingly, and in contrast to the closely related FLAP protein, LTC4 synthase is distributed on the outer nuclear membrane and in the peripheral ER but is strictly excluded from the inner nuclear membrane. Furthermore, the active site of LTC4 synthase is localized in the lumen of the nuclear envelope and ER. These results support the possibility that the intracellular synthesis of LTB4 and LTC4may be differentially compartmentalized and that LTC4 is synthesized on the luminal face of the ER membrane from where it must be returned to the cytoplasm for eventual export by MRP. In addition, the differential localization of two integral membrane proteins of essentially the same size, with a high degree of identity, indicates that mechanisms other than size-dependent exclusion regulate the passage of integral membrane proteins to the inner nuclear membrane.DISCUSSIONEndogenous or overexpressed LTC4 synthase show the same characteristic dense staining in the nuclear envelope extending to the peripheral ER (Fig. 2), which was clearly distinct from that of lamin A/C. The digital overlap with lamin showed a bright yellow rim surrounding the nucleus. Since lamin A/C was restricted to a tight nuclear rim, this suggested that the distribution of LTC4synthase extended well into the peripheral ER. This distribution was seen for FLAP in peripheral blood human monocytes (7Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heibein J.A. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Crossref PubMed Scopus (358) Google Scholar). When examined by EM, FLAP was distributed almost equally to the inner and outer nuclear membrane (7Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heibein J.A. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Crossref PubMed Scopus (358) Google Scholar). Furthermore, 5-LO, which was localized in the cytosol of resting monocytes and neutrophils, was associated with the outer and inner nuclear membrane after cell activation. This movement of 5-LO through the nuclear pore has been well characterized (23Brock T.G. McNish R.W. Peters-Golden M. J. Biol. Chem. 1995; 270: 21652-21658Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 24Chen X-S. Zhang Y-Y. Funk C.D. J. Biol. Chem. 1998; 273: 31237-31244Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and is mediated by a nuclear localization sequence at the C-terminal of the molecule (24Chen X-S. Zhang Y-Y. Funk C.D. J. Biol. Chem. 1998; 273: 31237-31244Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 25Lepley R.A. Fitzpatrick F.A. Arch. Biochem. Biophys. 1998; 356: 71-76Crossref PubMed Scopus (35) Google Scholar). Recently, LTA4 hydrolase has been shown to have the potential to move through nuclear pores and target to the nucleus in RBL cells (26Brock T.G. Maydanski E. McNish R.W. Peters-Golden M. J. Biol. Chem. 2001; 276: 35071-35077Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), indicating that LTB4 would be synthesized in the nuclear compartment. It was not determined whether LTA4 hydrolase was translocated to the cell membrane after activation.LTC4 synthase and FLAP share 52% identity between amino acids 41 and 97 of FLAP and amino acids 45–101 of LTC4synthase, which includes the two loops of the active site. Because of this high identity and the small 17-kDa size of these two proteins, it was highly surprising that the distribution of LTC4synthase between the inner and outer nuclear membrane was distinct from that of FLAP (Fig. 7). This restriction of LTC4 synthase to the outer nuclear membrane combined with the observation that LTB4 may be made within the nucleus suggests that the synthesis of LTB4 and LTC4 can be differentially compartmentalized within cells. This points to potential differences in their role in intracellular function and intracellular trafficking. Various studies have suggested a role for LTB4in gene transcription as a ligand for peroxisome proliferator-activated receptor γ (27Devchand P.R. Keller H. Peters J.M. Vazquez M. Gonzalez F.J. Wahli W. Nature. 1996; 384: 39-43Crossref PubMed Scopus (1199) Google Scholar). The exclusion of LTC4 synthase from the inner nuclear membrane suggests that LTC4 is less likely than LTB4 to play an intracellular role in modulating nuclear function and in transcriptional regulation.It is generally accepted that after their synthesis and insertion in the ER, integral membrane proteins become localized to the inner nuclear membrane by lateral diffusion through the proteolipid bilayer of the outer nuclear membrane followed by diffusion around the nuclear pore (28Soullam B. Worman H.J. J. Cell Biol. 1995; 130: 15-27Crossref PubMed Scopus (178) Google Scholar, 29Ellenberg J. Siggia E.D. Moreira J.E. Smith C.L. Presley J.F. Worman H.J. Lippincott-Schwartz J. J. Cell Biol. 1997; 138: 1193-1206Crossref PubMed Scopus (623) Google Scholar). In this model, the proteins are subsequently immobilized in the inner membrane by binding to immobile nucleoskeletal or nucleoplasmic ligands or by multimerization. The main mechanism excluding proteins from entry into the inner membrane is based on size, so that proteins with cytosolic/nucleoplasmic domains greater than 70 kDa fail to localize to the inner nuclear membrane (28Soullam B. Worman H.J. J. Cell Biol. 1995; 130: 15-27Crossref PubMed Scopus (178) Google Scholar). Proteins that do not fall into either category would be potentially free to diffuse between all contiguous membrane domains and be equally represented in the inner and outer nuclear membrane. FLAP fulfills these postulates and fits in the latter group, being equally represented in the inner and outer membrane (7Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heibein J.A. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Crossref PubMed Scopus (358) Google Scholar). LTC4 synthase contradicts them, being a small protein essentially the same size as FLAP but being excluded from the inner nuclear membrane. Thus, a different mechanism must exist that allows FLAP free entry into the inner nuclear membrane and excludes LTC4 synthase. This includes the possibility that the primary sequence of FLAP contains a signal that allows it entry to the inner membrane or that LTC4 synthase contains a sequence that signals its exclusion.The orientation of the N and C termini to the cytoplasm (Fig. 2) and the loops of the active site to the ER lumen (Figs. 4 and 5) support the model of membrane topology shown in Fig 1 F. As described above, the release of LTC4 is dependent on its export from cells by the MRP-1 protein, which translocates its substrates from the cytosol to the extracellular space (12Lam B.K. Owen Jr., W.F. Austen K.F. Soberman R.J. J. Biol. Chem. 1989; 264: 12885-12889Abstract Full Text PDF PubMed Google Scholar, 13Jedlitschky G. Buchholz U. Keppler D. Eur. J. Biochem. 1994; 220: 599-606Crossref PubMed Scopus (148) Google Scholar, 14Leir I. Jedlitschky G. Bucholz U. Cole S.P. Deeley R.G. Keppler D. J. Biol. Chem. 1994; 269: 27807-27810Abstract Full Text PDF PubMed Google Scholar, 15Wijnholds J. Evers R. van Leusden M.R. Mol C.A. Zaman G.J. Mayer U. Beijnen J.H. van der Valk M. P. Krimpenfort P. Borst P. Nat. Med. 1997; 3: 1275-1279Crossref PubMed Scopus (399) Google Scholar). LTC4 does not diffuse across membranes and must reenter the cytoplasmic compartment to be accessible to the MRP-1 protein. The mechanism by which this occurs is not known. LTC4 is formed at the luminal face of the ER, where GSH concentrations are 2–3 mm (30Hwang C. Sinskey A.J. Lodish H.F. Science. 1992; 257: 1496-1502Crossref PubMed Scopus (1569) Google Scholar). GSH within the ER has been suggested to play a role mostly as a redox buffer controlling the state of sulfhydryl bonds (31Lundström-Ljung J. Holmgren A. J. Biol. Chem. 1995; 270: 7822-7828Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Our data suggest that GSH in the ER can serve as an enzymatic substrate in additional reactions. Both prostaglandin H synthases have also been shown to have their active site oriented toward the ER lumen. As for prostaglandin endoperoxides generated by prostaglandin H synthase-1 and -2 (32Spencer A.G. Woods J.W. Arakawa T. Singer I.I. Smith W.L. J. Biol. Chem. 1998; 273: 9886-9893Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar), how LTC4 moves from the luminal surface to be accessible to intracellular transport and export by the MRP protein remains an open question. Leukotrienes (LTs)1 are proinflammatory products of arachidonic acid (AA) metabolism. LTC4, the parent sulfidopeptide LT, is converted to LTD4 by γ-glutamyl transpeptidase. LTC4 and LTD4 have been implicated in the pathogenesis of asthma, renal disease (1Lewis R.A. Austen K.F. Soberman R.J. N. Engl. J. Med. 1990; 323: 645-655Crossref PubMed Scopus (1166) Google Scholar), and dendritic cell trafficking (2Robbiani D.F. Finch R.A. Jager D. Muller W.A. Sartorelli A.C. Randolph G.J. Cell. 2000; 103: 757-768Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). LTC4is formed in four biochemical steps. First, cytoplasmic phospholipase A2 releases AA from phospholipids (3Clark J.D. Lin L.L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 6: 1043-1051Abstract Full Text PDF Scopus (1454) Google Scholar, 4Qiu Z.H. Gijon M.A. de Carvalho M.S. Spencer D.M. Leslie C.C. J. Biol. Chem. 1998; 273: 8203-8211Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Five-lipoxygenase-activating protein (FLAP) then mediates the interaction between five-lipoxygenase (5-LO) and AA (5Dixon R.A. Diehl R.E. Opas E. Rands E. Vickers P.J. Evans J.F. Gillard J.W. Miller D.K. Nature. 1990; 343: 282-284Crossref PubMed Scopus (647) Google Scholar, 6Miller D.K. Gillard J.W. Vickers P.J. Sadowski S. Leveille C. Mancini J.A. Charleson P. Dixon R.A. Ford-Hutchinson A.W. Fortin R. Nature. 1990; 343: 278-281Crossref PubMed Scopus (379) Google Scholar, 7Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heibein J.A. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Crossref PubMed Scopus (358) Google Scholar, 8Mancini J.A. Abromowitz M. Cox M.E. Wong E. Charleson S. Perrier H. Wang Z. Peptiboon P. Vickers P. FEBS Lett. 1993; 318: 277-281Crossref PubMed Scopus (180) Google Scholar) yielding both 5(S)-hydroperoxyeicosatetraenoic acid and LTA4 (9Rouzer C.A. Matsumoto T. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 857-861Crossref PubMed Scopus (268) Google Scholar). LTC4 synthase then catalyzes the conjugation of GSH with LTA4 to form LTC4 (10Penrose J.F. Gagnon L. Goppelt-Struebe M. Myers P. Lam B.K. Jack R.M. Austen K.F. Soberman R.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11603-11606Crossref PubMed Scopus (67) Google Scholar,11Lam B.K. Penrose J.F. Freeman G.F. Austen K.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7663-7667Crossref PubMed Scopus (247) Google Scholar). LTC4 is exported from cells by the plasma membrane protein MRP-1 (12Lam B.K. Owen Jr., W.F. Austen K.F. Soberman R.J. J. Biol. Chem. 1989; 264: 12885-12889Abstract Full Text PDF PubMed Google Scholar, 13Jedlitschky G. Buchholz U. Keppler D. Eur. J. Biochem. 1994; 220: 599-606Crossref PubMed Scopus (148) Google Scholar, 14Leir I. Jedlitschky G. Bucholz U. Cole S.P. Deeley R.G. Keppler D. J. Biol. Chem. 1994; 269: 27807-27810Abstract Full Text PDF PubMed Google Scholar), and cells from MRP-1 knockout mice do not export LTC4 (15Wijnholds J. Evers R. van Leusden M.R. Mol C.A. Zaman G.J. Mayer U. Beijnen J.H. van der Valk M. P. Krimpenfort P. Borst P. Nat. Med. 1997; 3: 1275-1279Crossref PubMed Scopus (399) Google Scholar). A major strategy used by cells to prevent the formation of these bioactive molecules under resting conditions is compartmentalization of the biosynthetic enzymes. In circulating, quiescent peripheral blood cells, both cytoplasmic phospholipase A2 and 5-LO are localized to the cytoplasmic compartment, whereas FLAP is localized to the inner and outer nuclear membrane, preventing the release of AA and the formation of LTs. When leukocytes and mast cells are activated, cytoplasmic phospholipase A2 translocates to the nuclear envelope. At the same time, 5-LO translocates to the inner and outer nuclear membranes. The association of 5-LO with these membranes is regulated, in part, by FLAP (7Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heibein J.A. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Crossref PubMed Scopus (358) Google Scholar, 16Glover S. Bayburt T. Jonas M. Chi E. Chi E. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15567Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). LTC4 synthase and FLAP are closely related 17-kDa transmembrane proteins, and both are predicted to have three hydrophobic domains. The region that extends from the first predicted hydrophilic loop to the third hydrophobic domain is highly homologous between the two proteins (Fig.1). The localization of LTC4 synthase has not been characterized in detail. FLAP is distributed between the inner and outer nuclear membranes and peripheral ER. The first hydrophilic loop of FLAP was localized to the lumen of the nuclear membrane (7Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heibein J.A. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Crossref PubMed Scopus (358) Google Scholar), but the topology of the second hydrophilic loop and the C and N termini of FLAP are unknown, although a model with three transmembrane domains was proposed (8Mancini J.A. Abromowitz M. Cox M.E. Wong E. Charleson S. Perrier H. Wang Z. Peptiboon P. Vickers P. FEBS Lett. 1993; 318: 277-281Crossref PubMed Scopus (180) Google Scholar) (Fig. 1 A). The first hydrophilic loop of LTC4 synthase binds LTA4 (8Mancini J.A. Abromowitz M. Cox M.E. Wong E. Charleson S. Perrier H. Wang Z. Peptiboon P. Vickers P. FEBS Lett. 1993; 318: 277-281Crossref PubMed Scopus (180) Google Scholar, 17Lam B.K. Penrose J.F. Xu K. Baldasaro M.H. Austen K.F. J. Biol. Chem. 1997; 272: 13923-13928Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). GSH conjugation is determined by amino acid residue Tyr93 in the second hydrophilic loop, suggesting that the hydrophilic loops are on the same, cytoplasmic side of the membrane (Fig. 1 B). No experimental determination of the topology has been made to support this model, and other relationships between the hydrophilic loops and the C-and N termini may exist (Fig.1, C–G). We have analyzed the distribution and topology of LTC4synthase using a combination of immunofluorescence, confocal, and electron microscopy combined with differential membrane permeabilization by streptolysin O (SLO). Surprisingly, and in contrast to the closely related FLAP protein, LTC4 synthase is distributed on the outer nuclear membrane and in the peripheral ER but is strictly excluded from the inner nuclear membrane. Furthermore, the active site of LTC4 synthase is localized in the lumen of the nuclear envelope and ER. These results support the possibility that the intracellular synthesis of LTB4 and LTC4may be differentially compartmentalized and that LTC4 is synthesized on the luminal face of the ER membrane from where it must be returned to the cytoplasm for eventual export by MRP. In addition, the differential localization of two integral membrane proteins of essentially the same size, with a high degree of identity, indicates that mechanisms other than size-dependent exclusion regulate the passage of integral membrane proteins to the inner nuclear membrane. DISCUSSIONEndogenous or overexpressed LTC4 synthase show the same characteristic dense staining in the nuclear envelope extending to the peripheral ER (Fig. 2), which was clearly distinct from that of lamin A/C. The digital overlap with lamin showed a bright yellow rim surrounding the nucleus. Since lamin A/C was restricted to a tight nuclear rim, this suggested that the distribution of LTC4synthase extended well into the peripheral ER. This distribution was seen for FLAP in peripheral blood human monocytes (7Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heibein J.A. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Crossref PubMed Scopus (358) Google Scholar). When examined by EM, FLAP was distributed almost equally to the inner and outer nuclear membrane (7Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heibein J.A. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Crossref PubMed Scopus (358) Google Scholar). Furthermore, 5-LO, which was localized in the cytosol of resting monocytes and neutrophils, was associated with the outer and inner nuclear membrane after cell activation. This movement of 5-LO through the nuclear pore has been well characterized (23Brock T.G. McNish R.W. Peters-Golden M. J. Biol. Chem. 1995; 270: 21652-21658Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 24Chen X-S. Zhang Y-Y. Funk C.D. J. Biol. Chem. 1998; 273: 31237-31244Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and is mediated by a nuclear localization sequence at the C-terminal of the molecule (24Chen X-S. Zhang Y-Y. Funk C.D. J. Biol. Chem. 1998; 273: 31237-31244Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 25Lepley R.A. Fitzpatrick F.A. Arch. Biochem. Biophys. 1998; 356: 71-76Crossref PubMed Scopus (35) Google Scholar). Recently, LTA4 hydrolase has been shown to have the potential to move through nuclear pores and target to the nucleus in RBL cells (26Brock T.G. Maydanski E. McNish R.W. Peters-Golden M. J. Biol. Chem. 2001; 276: 35071-35077Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), indicating that LTB4 would be synthesized in the nuclear compartment. It was not determined whether LTA4 hydrolase was translocated to the cell membrane after activation.LTC4 synthase and FLAP share 52% identity between amino acids 41 and 97 of FLAP and amino acids 45–101 of LTC4synthase, which includes the two loops of the active site. Because of this high identity and the small 17-kDa size of these two proteins, it was highly surprising that the distribution of LTC4synthase between the inner and outer nuclear membrane was distinct from that of FLAP (Fig. 7). This restriction of LTC4 synthase to the outer nuclear membrane combined with the observation that LTB4 may be made within the nucleus suggests that the synthesis of LTB4 and LTC4 can be differentially compartmentalized within cells. This points to potential differences in their role in intracellular function and intracellular trafficking. Various studies have suggested a role for LTB4in gene transcription as a ligand for peroxisome proliferator-activated receptor γ (27Devchand P.R. Keller H. Peters J.M. Vazquez M. Gonzalez F.J. Wahli W. Nature. 1996; 384: 39-43Crossref PubMed Scopus (1199) Google Scholar). The exclusion of LTC4 synthase from the inner nuclear membrane suggests that LTC4 is less likely than LTB4 to play an intracellular role in modulating nuclear function and in transcriptional regulation.It is generally accepted that after their synthesis and insertion in the ER, integral membrane proteins become localized to the inner nuclear membrane by lateral diffusion through the proteolipid bilayer of the outer nuclear membrane followed by diffusion around the nuclear pore (28Soullam B. Worman H.J. J. Cell Biol. 1995; 130: 15-27Crossref PubMed Scopus (178) Google Scholar, 29Ellenberg J. Siggia E.D. Moreira J.E. Smith C.L. Presley J.F. Worman H.J. Lippincott-Schwartz J. J. Cell Biol. 1997; 138: 1193-1206Crossref PubMed Scopus (623) Google Scholar). In this model, the proteins are subsequently immobilized in the inner membrane by binding to immobile nucleoskeletal or nucleoplasmic ligands or by multimerization. The main mechanism excluding proteins from entry into the inner membrane is based on size, so that proteins with cytosolic/nucleoplasmic domains greater than 70 kDa fail to localize to the inner nuclear membrane (28Soullam B. Worman H.J. J. Cell Biol. 1995; 130: 15-27Crossref PubMed Scopus (178) Google Scholar). Proteins that do not fall into either category would be potentially free to diffuse between all contiguous membrane domains and be equally represented in the inner and outer nuclear membrane. FLAP fulfills these postulates and fits in the latter group, being equally represented in the inner and outer membrane (7Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heibein J.A. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Crossref PubMed Scopus (358) Google Scholar). LTC4 synthase contradicts them, being a small protein essentially the same size as FLAP but being excluded from the inner nuclear membrane. Thus, a different mechanism must exist that allows FLAP free entry into the inner nuclear membrane and excludes LTC4 synthase. This includes the possibility that the primary sequence of FLAP contains a signal that allows it entry to the inner membrane or that LTC4 synthase contains a sequence that signals its exclusion.The orientation of the N and C termini to the cytoplasm (Fig. 2) and the loops of the active site to the ER lumen (Figs. 4 and 5) support the model of membrane topology shown in Fig 1 F. As described above, the release of LTC4 is dependent on its export from cells by the MRP-1 protein, which translocates its substrates from the cytosol to the extracellular space (12Lam B.K. Owen Jr., W.F. Austen K.F. Soberman R.J. J. Biol. Chem. 1989; 264: 12885-12889Abstract Full Text PDF PubMed Google Scholar, 13Jedlitschky G. Buchholz U. Keppler D. Eur. J. Biochem. 1994; 220: 599-606Crossref PubMed Scopus (148) Google Scholar, 14Leir I. Jedlitschky G. Bucholz U. Cole S.P. Deeley R.G. Keppler D. J. Biol. Chem. 1994; 269: 27807-27810Abstract Full Text PDF PubMed Google Scholar, 15Wijnholds J. Evers R. van Leusden M.R. Mol C.A. Zaman G.J. Mayer U. Beijnen J.H. van der Valk M. P. Krimpenfort P. Borst P. Nat. Med. 1997; 3: 1275-1279Crossref PubMed Scopus (399) Google Scholar). LTC4 does not diffuse across membranes and must reenter the cytoplasmic compartment to be accessible to the MRP-1 protein. The mechanism by which this occurs is not known. LTC4 is formed at the luminal face of the ER, where GSH concentrations are 2–3 mm (30Hwang C. Sinskey A.J. Lodish H.F. Science. 1992; 257: 1496-1502Crossref PubMed Scopus (1569) Google Scholar). GSH within the ER has been suggested to play a role mostly as a redox buffer controlling the state of sulfhydryl bonds (31Lundström-Ljung J. Holmgren A. J. Biol. Chem. 1995; 270: 7822-7828Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Our data suggest that GSH in the ER can serve as an enzymatic substrate in additional reactions. Both prostaglandin H synthases have also been shown to have their active site oriented toward the ER lumen. As for prostaglandin endoperoxides generated by prostaglandin H synthase-1 and -2 (32Spencer A.G. Woods J.W. Arakawa T. Singer I.I. Smith W.L. J. Biol. Chem. 1998; 273: 9886-9893Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar), how LTC4 moves from the luminal surface to be accessible to intracellular transport and export by the MRP protein remains an open question. Endogenous or overexpressed LTC4 synthase show the same characteristic dense staining in the nuclear envelope extending to the peripheral ER (Fig. 2), which was clearly distinct from that of lamin A/C. The digital overlap with lamin showed a bright yellow rim surrounding the nucleus. Since lamin A/C was restricted to a tight nuclear rim, this suggested that the distribution of LTC4synthase extended well into the peripheral ER. This distribution was seen for FLAP in peripheral blood human monocytes (7Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heibein J.A. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Crossref PubMed Scopus (358) Google Scholar). When examined by EM, FLAP was distributed almost equally to the inner and outer nuclear membrane (7Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heibein J.A. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Crossref PubMed Scopus (358) Google Scholar). Furthermore, 5-LO, which was localized in the cytosol of resting monocytes and neutrophils, was associated with the outer and inner nuclear membrane after cell activation. This movement of 5-LO through the nuclear pore has been well characterized (23Brock T.G. McNish R.W. Peters-Golden M. J. Biol. Chem. 1995; 270: 21652-21658Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 24Chen X-S. Zhang Y-Y. Funk C.D. J. Biol. Chem. 1998; 273: 31237-31244Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and is mediated by a nuclear localization sequence at the C-terminal of the molecule (24Chen X-S. Zhang Y-Y. Funk C.D. J. Biol. Chem. 1998; 273: 31237-31244Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 25Lepley R.A. Fitzpatrick F.A. Arch. Biochem. Biophys. 1998; 356: 71-76Crossref PubMed Scopus (35) Google Scholar). Recently, LTA4 hydrolase has been shown to have the potential to move through nuclear pores and target to the nucleus in RBL cells (26Brock T.G. Maydanski E. McNish R.W. Peters-Golden M. J. Biol. Chem. 2001; 276: 35071-35077Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), indicating that LTB4 would be synthesized in the nuclear compartment. It was not determined whether LTA4 hydrolase was translocated to the cell membrane after activation. LTC4 synthase and FLAP share 52% identity between amino acids 41 and 97 of FLAP and amino acids 45–101 of LTC4synthase, which includes the two loops of the active site. Because of this high identity and the small 17-kDa size of these two proteins, it was highly surprising that the distribution of LTC4synthase between the inner and outer nuclear membrane was distinct from that of FLAP (Fig. 7). This restriction of LTC4 synthase to the outer nuclear membrane combined with the observation that LTB4 may be made within the nucleus suggests that the synthesis of LTB4 and LTC4 can be differentially compartmentalized within cells. This points to potential differences in their role in intracellular function and intracellular trafficking. Various studies have suggested a role for LTB4in gene transcription as a ligand for peroxisome proliferator-activated receptor γ (27Devchand P.R. Keller H. Peters J.M. Vazquez M. Gonzalez F.J. Wahli W. Nature. 1996; 384: 39-43Crossref PubMed Scopus (1199) Google Scholar). The exclusion of LTC4 synthase from the inner nuclear membrane suggests that LTC4 is less likely than LTB4 to play an intracellular role in modulating nuclear function and in transcriptional regulation. It is generally accepted that after their synthesis and insertion in the ER, integral membrane proteins become localized to the inner nuclear membrane by lateral diffusion through the proteolipid bilayer of the outer nuclear membrane followed by diffusion around the nuclear pore (28Soullam B. Worman H.J. J. Cell Biol. 1995; 130: 15-27Crossref PubMed Scopus (178) Google Scholar, 29Ellenberg J. Siggia E.D. Moreira J.E. Smith C.L. Presley J.F. Worman H.J. Lippincott-Schwartz J. J. Cell Biol. 1997; 138: 1193-1206Crossref PubMed Scopus (623) Google Scholar). In this model, the proteins are subsequently immobilized in the inner membrane by binding to immobile nucleoskeletal or nucleoplasmic ligands or by multimerization. The main mechanism excluding proteins from entry into the inner membrane is based on size, so that proteins with cytosolic/nucleoplasmic domains greater than 70 kDa fail to localize to the inner nuclear membrane (28Soullam B. Worman H.J. J. Cell Biol. 1995; 130: 15-27Crossref PubMed Scopus (178) Google Scholar). Proteins that do not fall into either category would be potentially free to diffuse between all contiguous membrane domains and be equally represented in the inner and outer nuclear membrane. FLAP fulfills these postulates and fits in the latter group, being equally represented in the inner and outer membrane (7Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heibein J.A. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Crossref PubMed Scopus (358) Google Scholar). LTC4 synthase contradicts them, being a small protein essentially the same size as FLAP but being excluded from the inner nuclear membrane. Thus, a different mechanism must exist that allows FLAP free entry into the inner nuclear membrane and excludes LTC4 synthase. This includes the possibility that the primary sequence of FLAP contains a signal that allows it entry to the inner membrane or that LTC4 synthase contains a sequence that signals its exclusion. The orientation of the N and C termini to the cytoplasm (Fig. 2) and the loops of the active site to the ER lumen (Figs. 4 and 5) support the model of membrane topology shown in Fig 1 F. As described above, the release of LTC4 is dependent on its export from cells by the MRP-1 protein, which translocates its substrates from the cytosol to the extracellular space (12Lam B.K. Owen Jr., W.F. Austen K.F. Soberman R.J. J. Biol. Chem. 1989; 264: 12885-12889Abstract Full Text PDF PubMed Google Scholar, 13Jedlitschky G. Buchholz U. Keppler D. Eur. J. Biochem. 1994; 220: 599-606Crossref PubMed Scopus (148) Google Scholar, 14Leir I. Jedlitschky G. Bucholz U. Cole S.P. Deeley R.G. Keppler D. J. Biol. Chem. 1994; 269: 27807-27810Abstract Full Text PDF PubMed Google Scholar, 15Wijnholds J. Evers R. van Leusden M.R. Mol C.A. Zaman G.J. Mayer U. Beijnen J.H. van der Valk M. P. Krimpenfort P. Borst P. Nat. Med. 1997; 3: 1275-1279Crossref PubMed Scopus (399) Google Scholar). LTC4 does not diffuse across membranes and must reenter the cytoplasmic compartment to be accessible to the MRP-1 protein. The mechanism by which this occurs is not known. LTC4 is formed at the luminal face of the ER, where GSH concentrations are 2–3 mm (30Hwang C. Sinskey A.J. Lodish H.F. Science. 1992; 257: 1496-1502Crossref PubMed Scopus (1569) Google Scholar). GSH within the ER has been suggested to play a role mostly as a redox buffer controlling the state of sulfhydryl bonds (31Lundström-Ljung J. Holmgren A. J. Biol. Chem. 1995; 270: 7822-7828Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Our data suggest that GSH in the ER can serve as an enzymatic substrate in additional reactions. Both prostaglandin H synthases have also been shown to have their active site oriented toward the ER lumen. As for prostaglandin endoperoxides generated by prostaglandin H synthase-1 and -2 (32Spencer A.G. Woods J.W. Arakawa T. Singer I.I. Smith W.L. J. Biol. Chem. 1998; 273: 9886-9893Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar), how LTC4 moves from the luminal surface to be accessible to intracellular transport and export by the MRP protein remains an open question. We thank Dr. Sylvie Breton (Massachusetts General Hospital Program in Membrane Biology) for assistance with confocal microscopy." @default.
W2028881470 created "2016-06-24" @default.
W2028881470 creator A5011167573 @default.
W2028881470 creator A5023258176 @default.
W2028881470 creator A5041377113 @default.
W2028881470 creator A5085550092 @default.
W2028881470 creator A5087672907 @default.
W2028881470 date "2002-08-01" @default.
W2028881470 modified "2023-09-28" @default.
W2028881470 title "Membrane Localization and Topology of Leukotriene C4 Synthase" @default.
W2028881470 cites W1499727792 @default.
W2028881470 cites W1502426706 @default.
W2028881470 cites W1647167187 @default.
W2028881470 cites W1671834350 @default.
W2028881470 cites W1975793827 @default.
W2028881470 cites W1980832959 @default.
W2028881470 cites W1982407164 @default.
W2028881470 cites W1982732431 @default.
W2028881470 cites W1985032385 @default.
W2028881470 cites W2000825465 @default.
W2028881470 cites W2007082033 @default.
W2028881470 cites W2014768234 @default.
W2028881470 cites W2019328429 @default.
W2028881470 cites W2034058468 @default.
W2028881470 cites W2038575244 @default.
W2028881470 cites W2039376197 @default.
W2028881470 cites W2040062751 @default.
W2028881470 cites W2041159500 @default.
W2028881470 cites W2050994990 @default.
W2028881470 cites W2051958752 @default.
W2028881470 cites W2052614246 @default.
W2028881470 cites W2056204193 @default.
W2028881470 cites W2062080431 @default.
W2028881470 cites W2064700667 @default.
W2028881470 cites W2077794392 @default.
W2028881470 cites W2079014952 @default.
W2028881470 cites W2097453018 @default.
W2028881470 cites W2114869075 @default.
W2028881470 cites W2121737195 @default.
W2028881470 cites W2143350004 @default.
W2028881470 cites W2145716903 @default.
W2028881470 cites W2171991181 @default.
W2028881470 doi "https://doi.org/10.1074/jbc.m203074200" @default.
W2028881470 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12023288" @default.
W2028881470 hasPublicationYear "2002" @default.
W2028881470 type Work @default.
W2028881470 sameAs 2028881470 @default.
W2028881470 citedByCount "53" @default.
W2028881470 countsByYear W20288814702012 @default.
W2028881470 countsByYear W20288814702016 @default.
W2028881470 countsByYear W20288814702018 @default.
W2028881470 countsByYear W20288814702019 @default.
W2028881470 countsByYear W20288814702020 @default.
W2028881470 countsByYear W20288814702021 @default.
W2028881470 countsByYear W20288814702022 @default.
W2028881470 crossrefType "journal-article" @default.
W2028881470 hasAuthorship W2028881470A5011167573 @default.
W2028881470 hasAuthorship W2028881470A5023258176 @default.
W2028881470 hasAuthorship W2028881470A5041377113 @default.
W2028881470 hasAuthorship W2028881470A5085550092 @default.
W2028881470 hasAuthorship W2028881470A5087672907 @default.
W2028881470 hasBestOaLocation W20288814701 @default.
W2028881470 hasConcept C112243037 @default.
W2028881470 hasConcept C114614502 @default.
W2028881470 hasConcept C144647389 @default.
W2028881470 hasConcept C181199279 @default.
W2028881470 hasConcept C184720557 @default.
W2028881470 hasConcept C185592680 @default.
W2028881470 hasConcept C203014093 @default.
W2028881470 hasConcept C2776042228 @default.
W2028881470 hasConcept C2776452501 @default.
W2028881470 hasConcept C2779543280 @default.
W2028881470 hasConcept C33923547 @default.
W2028881470 hasConcept C41625074 @default.
W2028881470 hasConcept C534586 @default.
W2028881470 hasConcept C55493867 @default.
W2028881470 hasConcept C86803240 @default.
W2028881470 hasConcept C95444343 @default.
W2028881470 hasConceptScore W2028881470C112243037 @default.
W2028881470 hasConceptScore W2028881470C114614502 @default.
W2028881470 hasConceptScore W2028881470C144647389 @default.
W2028881470 hasConceptScore W2028881470C181199279 @default.
W2028881470 hasConceptScore W2028881470C184720557 @default.
W2028881470 hasConceptScore W2028881470C185592680 @default.
W2028881470 hasConceptScore W2028881470C203014093 @default.
W2028881470 hasConceptScore W2028881470C2776042228 @default.
W2028881470 hasConceptScore W2028881470C2776452501 @default.
W2028881470 hasConceptScore W2028881470C2779543280 @default.
W2028881470 hasConceptScore W2028881470C33923547 @default.
W2028881470 hasConceptScore W2028881470C41625074 @default.
W2028881470 hasConceptScore W2028881470C534586 @default.
W2028881470 hasConceptScore W2028881470C55493867 @default.
W2028881470 hasConceptScore W2028881470C86803240 @default.
W2028881470 hasConceptScore W2028881470C95444343 @default.
W2028881470 hasIssue "32" @default.
W2028881470 hasLocation W20288814701 @default.
W2028881470 hasOpenAccess W2028881470 @default.
W2028881470 hasPrimaryLocation W20288814701 @default.