CERT Mediates Intermembrane Transfer of Various Molecular Species of Ceramides
2005; Elsevier BV; Volume: 280; Issue: 8 Linguagem: Inglês
10.1074/jbc.m409290200
ISSN1083-351X
AutoresKeigo Kumagai, Satoshi Yasuda, Kazuo Okemoto, Masahiro Nishijima, Shū Kobayashi, Kentaro Hanada,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoCeramide produced at the endoplasmic reticulum is transported to the Golgi apparatus for conversion to sphingomyelin. The main pathway of endoplasmic reticulum-to-Golgi transport of ceramide is mediated by CERT, a cytosolic 68-kDa protein, in a nonvesicular manner. CERT contains a domain that catalyzes the intermembrane transfer of natural C16-ceramide. In this study, we examined the ligand specificity of CERT in detail by using a cell-free assay system for intermembrane transfer of lipids. CERT did not mediate the transfer of sphingosine or sphingomyelin at all. The activity of CERT to transfer saturated and unsaturated diacylglycerols, which structurally resemble ceramide, was 5–10% of the activity toward C16-ceramide. Among four stereoisomers of C16-ceramide, CERT specifically recognized the natural d-erythro isomer. CERT efficiently transferred ceramides having C14, C16, C18, and C20 chains, but not longer acyl chains, and also mediated efficient transfer of C16-dihydroceramide and C16-phyto-ceramide. Binding assays showed that CERT also recognizes short chain fluorescent analogs of ceramide with a stoichiometry of 1:1. Moreover, (1R,3R)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecamide, which inhibited the CERT-dependent pathway of ceramide trafficking in intact cells, was found to be an antagonist of the CERT protein. These results indicate that CERT can mediate transfer of various types of ceramides that naturally exist and their close relatives. Ceramide produced at the endoplasmic reticulum is transported to the Golgi apparatus for conversion to sphingomyelin. The main pathway of endoplasmic reticulum-to-Golgi transport of ceramide is mediated by CERT, a cytosolic 68-kDa protein, in a nonvesicular manner. CERT contains a domain that catalyzes the intermembrane transfer of natural C16-ceramide. In this study, we examined the ligand specificity of CERT in detail by using a cell-free assay system for intermembrane transfer of lipids. CERT did not mediate the transfer of sphingosine or sphingomyelin at all. The activity of CERT to transfer saturated and unsaturated diacylglycerols, which structurally resemble ceramide, was 5–10% of the activity toward C16-ceramide. Among four stereoisomers of C16-ceramide, CERT specifically recognized the natural d-erythro isomer. CERT efficiently transferred ceramides having C14, C16, C18, and C20 chains, but not longer acyl chains, and also mediated efficient transfer of C16-dihydroceramide and C16-phyto-ceramide. Binding assays showed that CERT also recognizes short chain fluorescent analogs of ceramide with a stoichiometry of 1:1. Moreover, (1R,3R)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecamide, which inhibited the CERT-dependent pathway of ceramide trafficking in intact cells, was found to be an antagonist of the CERT protein. These results indicate that CERT can mediate transfer of various types of ceramides that naturally exist and their close relatives. The intracellular transport of lipids from the sites of their synthesis to their appropriate destinations must occur, because various steps in lipid biosynthesis occur in different intracellular compartments. The trafficking of integral membrane proteins in eukaryotic cells is mediated by transport vesicles, which load the desired set of proteins and deliver them to the correct organelles. By contrast, many types of lipid synthesized in the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; CHO, Chinese hamster ovary; StAR, steroidogenic acute regulatory protein; START, StAR-related lipid transfer; HPA-12, N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecamide; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; SCDase, sphingolipid ceramide N-deacylase; TBS, Tris-buffered saline; LPS, lipopolysaccharide; DMB, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene; NBD, 7-nitrobenzo-2-oxa-1,3-diazole. 1The abbreviations used are: ER, endoplasmic reticulum; CHO, Chinese hamster ovary; StAR, steroidogenic acute regulatory protein; START, StAR-related lipid transfer; HPA-12, N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecamide; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; SCDase, sphingolipid ceramide N-deacylase; TBS, Tris-buffered saline; LPS, lipopolysaccharide; DMB, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene; NBD, 7-nitrobenzo-2-oxa-1,3-diazole. have been suggested to be sorted to other organelles by nonvesicular mechanisms, although some lipid flux routes such as the endocytosis of plasma membrane lipids occur by vesicle-mediated mechanisms (1van Meer G. Holthuis J.C. Biochim. Biophys. Acta. 2000; 1486: 145-170Crossref PubMed Scopus (132) Google Scholar, 2Maxfield F.R. Wustner D. J. Clin. Investig. 2002; 110: 891-898Crossref PubMed Scopus (276) Google Scholar, 3Voelker D.R. Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins, and Membranes. 4th Ed. Elsevier Science Publishers B.V., Amsterdam, The Netherlands1991: 489-523Google Scholar). In mammalian cells, ceramide is synthesized at the ER and translocated to the Golgi compartment for conversion to sphingomyelin (4Merrill Jr., A.H. Jones D.D. Biochim. Biophys. Acta. 1990; 1044: 1-12Crossref PubMed Scopus (394) Google Scholar). There are at least two pathways by which ceramide is transported from the ER to the Golgi site for the synthesis of sphingomyelin: an ATP- and cytosol-dependent major pathway and an ATP- or cytosol-independent (or less dependent) minor pathway (5Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 6Fukasawa M. Nishijima M. Hanada K. J. Cell Biol. 1999; 144: 673-685Crossref PubMed Scopus (148) Google Scholar, 7Funakoshi T. Yasuda S. Fukasawa M. Nishijima M. Hanada K. J. Biol. Chem. 2000; 275: 29938-29945Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The major pathway is impaired in a Chinese hamster ovary (CHO) mutant cell line, LY-A, without any deficiency in the ER-to-Golgi transport of proteins (5Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 6Fukasawa M. Nishijima M. Hanada K. J. Cell Biol. 1999; 144: 673-685Crossref PubMed Scopus (148) Google Scholar, 7Funakoshi T. Yasuda S. Fukasawa M. Nishijima M. Hanada K. J. Biol. Chem. 2000; 275: 29938-29945Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). We have identified CERT as a factor defective in LY-A cells by functional rescue experiments, and we have shown that CERT mediates the ATP-dependent pathway of ER-to-Golgi trafficking of ceramide in a nonvesicular manner (8Hanada K. Kumagai K. Yasuda S. Miura Y. Kawano M. Fukasawa M. Nishijima M. Nature. 2003; 426: 803-809Crossref PubMed Scopus (815) Google Scholar).CERT is a tripartite cytosolic protein ∼600 amino acids in length (8Hanada K. Kumagai K. Yasuda S. Miura Y. Kawano M. Fukasawa M. Nishijima M. Nature. 2003; 426: 803-809Crossref PubMed Scopus (815) Google Scholar, 9Raya A. Revert F. Navarro S. Saus J. J. Biol. Chem. 1999; 274: 12642-12649Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 10Raya A. Revert-Ros F. Martinez-Martinez P. Navarro S. Rosello E. Vieites B. Granero F. Forteza J. Saus J. J. Biol. Chem. 2000; 275: 40392-40399Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The amino-terminal region of ∼120 amino acids is a phosphatidylinositol 4-phosphate (PtdIns4P)-binding pleckstrin homology domain, which can target the Golgi apparatus (11Levine T.P. Munro S. Curr. Biol. 2002; 12: 695-704Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). The next region of ∼250 amino acids (referred to as the middle region) contains coiled-coil motifs (9Raya A. Revert F. Navarro S. Saus J. J. Biol. Chem. 1999; 274: 12642-12649Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), which might play a role in homo- or hetero-oligomerization, and a motif that may participate in association with the ER (12Loewen C.J. Roy A. Levine T.P. EMBO J. 2003; 22: 2025-2035Crossref PubMed Scopus (437) Google Scholar). The carboxyl terminus of ∼230 amino acids is a steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain.START domains were initially recognized as putative lipid-binding domains of ∼210 amino acid residues, which exist in various types of proteins implicated in intracellular lipid transport, lipid metabolism, and signal transduction (13Ponting C.P. Aravind L. Trends Biochem. Sci. 1999; 24: 130-132Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 14Tsujishita Y. Hurley J.H. Nat. Struct. Biol. 2000; 7: 408-414Crossref PubMed Scopus (436) Google Scholar). Although more than 200 proteins have been nominated so far as proteins having START domains in data bases (for example, see smart.embl-heidelberg.de/), only a few have been experimentally shown to bind or transfer specific lipids. For example, StAR and MLN64 proteins recognize cholesterol (14Tsujishita Y. Hurley J.H. Nat. Struct. Biol. 2000; 7: 408-414Crossref PubMed Scopus (436) Google Scholar, 15Kallen C.B. Billheimer J.T. Summers S.A. Stayrook S.E. Lewis M. Strauss III, J.F. J. Biol. Chem. 1998; 273: 26285-26288Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 16Petrescu A.D. Gallegos A.M. Okamura Y. Strauss III, J.F. Schroeder F. J. Biol. Chem. 2001; 276: 36970-36982Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 17Zhang M. Liu P. Dwyer N.K. Christenson L.K. Fujimoto T. Martinez F. Comly M. Hanover J.A. Blanchette-Mackie E.J. Strauss III, J.F. J. Biol. Chem. 2002; 277: 33300-33310Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), and phosphatidylcholine (PtdCho)-transfer protein is capable of intermembrane transfer of PtdCho in vitro (18de Brouwer A.P. Bouma B. van Tiel C.M. Heerma W. Brouwers J.F. Bevers L.E. Westerman J. Roelofsen B. Wirtz K.W. Chem. Phys. Lipids. 2001; 112: 109-119Crossref PubMed Scopus (16) Google Scholar, 19Feng L. Cohen D.E. J. Lipid Res. 1998; 39: 1862-1869Abstract Full Text Full Text PDF PubMed Google Scholar). The silkworm Bombyx mori larvae produce a carotenoid-binding START domain (20Tabunoki H. Sugiyama H. Tanaka Y. Fujii H. Banno Y. Jouni Z.E. Kobayashi M. Sato R. Maekawa H. Tsuchida K. J. Biol. Chem. 2002; 277: 32133-32140Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). We demonstrated previously that the START domain of CERT can efficiently extract natural long chain C16-ceramide but not other types of lipids, including sphingosine, sphingomyelin, PtdCho, and cholesterol, from phospholipid bilayers (8Hanada K. Kumagai K. Yasuda S. Miura Y. Kawano M. Fukasawa M. Nishijima M. Nature. 2003; 426: 803-809Crossref PubMed Scopus (815) Google Scholar). We have also shown that the START domain of CERT greatly facilitated intermembrane transfer of C16-ceramide in a cell-free system (8Hanada K. Kumagai K. Yasuda S. Miura Y. Kawano M. Fukasawa M. Nishijima M. Nature. 2003; 426: 803-809Crossref PubMed Scopus (815) Google Scholar). However, many details as to the substrate specificity of the START domain of CERT remain undetermined, although various molecular species of ceramide exist in mammalian cells.In most types of mammalian cells, the hydrophobic moiety of complex sphingolipids is mainly composed of ceramide but also includes dihydroceramide and phytoceramide at low levels (21Ramstedt B. Leppimaki P. Axberg M. Slotte J.P. Eur. J. Biochem. 1999; 266: 997-1002Crossref PubMed Scopus (73) Google Scholar, 22Karlsson K.A. Samuelsson B.E. Steen G.O. Biochim. Biophys. Acta. 1973; 316: 336-362Crossref PubMed Scopus (52) Google Scholar, 23Crossman M.W. Hirschberg C.B. J. Biol. Chem. 1977; 252: 5815-5819Abstract Full Text PDF PubMed Google Scholar). Notably, dihydrosphingomyelin is abundant in human lens membranes (24Yappert M.C. Borchman D. Chem. Phys. Lipids. 2004; 129: 1-20Crossref PubMed Scopus (69) Google Scholar, 25Byrdwell W.C. Borchman D. Ophthalmic Res. 1997; 29: 191-206Crossref PubMed Scopus (55) Google Scholar). Moreover, the length of the amido acyl chain of the ceramide moiety is diverse; C16-C26 acyl chains are observed in natural sphingomyelin. Notably, C18- and C24:1-ceramide is predominant for sphingomyelin in the brain (21Ramstedt B. Leppimaki P. Axberg M. Slotte J.P. Eur. J. Biochem. 1999; 266: 997-1002Crossref PubMed Scopus (73) Google Scholar, 26O'Brien J.S. Rouser G. J. Lipid Res. 1964; 5: 339-342Abstract Full Text PDF PubMed Google Scholar, 27Raghaven S. Spielvogel C. Kanfer J.N. Lipids. 1973; 8: 517-521Crossref PubMed Scopus (7) Google Scholar), whereas C16-ceramide is predominant in many other tissues (22Karlsson K.A. Samuelsson B.E. Steen G.O. Biochim. Biophys. Acta. 1973; 316: 336-362Crossref PubMed Scopus (52) Google Scholar, 28Taketomi T. Kawamura N. J. Biochem. (Tokyo). 1972; 72: 791-798Crossref PubMed Scopus (18) Google Scholar, 29Dobrzyn A. Gorski J. Am. J. Physiol. 2002; 282: E277-E285Crossref PubMed Scopus (94) Google Scholar, 30Fex G. Biochim. Biophys. Acta. 1971; 231: 161-169Crossref PubMed Scopus (18) Google Scholar). Such structural diversity in the ceramide moiety may affect the nature of membranes where complex sphingolipids are abundant. The physiological importance of the diversity of the ceramide structure has also been recognized, based on differences in bio-modulation activity between ceramide and dihydroceramide (31Bielawska A. Crane H.M. Liotta D. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1993; 268: 26226-26232Abstract Full Text PDF PubMed Google Scholar) or between natural long chain ceramide and unnatural short chain ceramide (32van Blitterswijk W.J. van der Luit A.H. Veldman R.J. Verheij M. Borst J. Biochem. J. 2003; 369: 199-211Crossref PubMed Scopus (380) Google Scholar). Hence, it should be of biological significance to determine whether CERT can catalyze the intermembrane transfer of various species of ceramide and its relatives in addition to C16-ceramide.In the present study, we show that CERT is capable of mediating the intermembrane transfer of various types of ceramides that naturally exist in its START domain-dependent manner. In addition, we show that an inhibitor of ER-to-Golgi transport of ceramide is an antagonist of CERT.EXPERIMENTAL PROCEDURESMaterials—Palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, nervonic acid, d-ribo-phytosphingosine, and Clostridium perfringens phospholipase C were purchased from Sigma. d-erythro-Sphingosine, d-threo-sphingosine, l-erythro-sphingosine, l-threo-sphingosine, d-erythro-dihydrosphingosine, and porcine-derived lactosylceramide were from Matreya Inc., and egg phosphatidylcholine (PtdCho) and egg phosphatidylethanolamine (PtdEtn) were from Avanti Polar Lipids Inc. N-[palmitoyl-1-14C]Palmitoyl-d-erythro-sphingosine (55 mCi/mmol), [1-14C]palmitic acid (55 mCi/mmol), [oleoyl-1-14C]dioleoyl-rac-glycerol (55 mCi/mmol), l-α-[choline-methyl-14C]-dipalmitoyl-PtdCho (55 mCi/mmol), [choline-methyl-14C]sphingomyelin (55 mCi/mmol), l-α-[dipalmitoyl-1-14C]dipalmitoyl-PtdCho (100 mCi/mmol), and l-α-[2-palmitoyl-9,10-3H]dipalmitoyl-PtdCho (40 Ci/mmol) were from American Radiolabeled Chemicals. d-erythro-[3-3H]Sphingosine (23 Ci/mmol) was from PerkinElmer Life Sciences, and [1,2-3H]-cholesterol (49 Ci/mmol) was from Amersham Biosciences. TLC plates (Silica Gel 60) were from Merck, and TALON metal affinity resin was from Clontech. Ricinus communis lectin was from Honen Inc. (Tokyo), and sphingolipid ceramide N-deacylase (SCDase) was from Takara Bio Inc. BODIPY® FL C5-ceramide or N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C5-DMB-ceramide), 6-[N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]caproyl-d-erythro-sphingosine (C6-NBD-ceramide), and lipopolysaccharide (LPS) Alexa Fluor® 488 were purchased from Invitrogen. (1R,3R)-N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecamide (HPA-12) and its derivatives were chemically synthesized as described (33Nakamura Y. Matsubara R. Kitagawa H. Kobayashi S. Kumagai K. Yasuda S. Hanada K. J. Med. Chem. 2003; 46: 3688-3695Crossref PubMed Scopus (41) Google Scholar).Preparation of C16-d-Erythro Types of [14C]Ceramide, [14C]Dihydroceramide, and [14C]Phytoceramide and Unnatural Stereoisomers of C16-[14C]Ceramide by Fatty Acid Transfer Reactions with SCDase—The structures of synthesized lipids and the yields of the synthesis are listed in Fig. 1. Natural stereoisomers of C16-[14C]ceramide, C16-[14C]dihydroceramide, and C16-[14C]phytoceramide and unnatural stereoisomers of C16-[14C]ceramide were synthesized by adaptation of the SCDase-catalyzing fatty acid transfer reaction (34Mitsutake S. Kita K. Okino N. Ito M. Anal. Biochem. 1997; 247: 52-57Crossref PubMed Scopus (46) Google Scholar). In our standard method, 400 nmol of [14C]palmitic acid (55 mCi/mmol) and 2 μmol of appropriate sphingoid base (d-erythro-sphingosine, d-threo-sphingosine, l-erythro-sphingosine, l-threo-sphingosine, d-erythro-dihydrosphingosine, or d-ribo-phytosphingosine) dissolved in ethanol were mixed in a screw-capped Pyrex glass tube and dried under nitrogen gas stream. After adding 2 ml of buffer A (50 mm sodium phosphate buffer (pH 7.0) containing 0.2% Triton X-100) to the dried lipids, the tube was sonicated with a bath-type sonicator for 5 min. 2 ml of SCDase (1 milliunit/ml in buffer A) was then added to the tube, and the mixture was incubated at 37 °C for ∼16 h. Lipids were extracted from the mixture by the method of Bligh and Dyer (35Bligh E.G. Dyer W.J. Can. J. Med. Sci. 1959; 37: 911-917Google Scholar), dried, dissolved in 400 μl of chloroform/methanol (19:1, by volume), and subjected to TLC with a solvent system of benzene, diethyl ether, ethyl acetate, methanol, 25% (w/v) ammonia (65:7.5:7.5:25:0.75, v/v). After detection of radioactive lipids separated on the TLC plate with a BAS1800 image analyzer (Fuji Film), gel containing a desired radioactive lipid was collected from the TLC plate by scraping. By this image analysis, the efficiency of conversion of the radiolabeled starting material to its ceramide product by the SCDase reaction was also estimated. For extraction of the radioactive lipid by the method of Bligh and Dyer (35Bligh E.G. Dyer W.J. Can. J. Med. Sci. 1959; 37: 911-917Google Scholar), 1.9 ml of a solvent of 0.1 m KCl/chloroform/methanol (0.8:1:2, v/v) was added to the collected gel and mixed with a swirling mixer. After addition of 0.5 ml of chloroform and 0.5 ml of 0.1 m KCl to the mixture for phase separation, the mixture was centrifuged (1000 × g, 3 min), and the lower organic phase was collected as a lipid extract. Then the extract was dried under a nitrogen gas stream, dissolved in 1 ml of chloroform/methanol (19:1,v/v), and stored at –20 °C. The radioactivities of synthesized radioactive lipids were determined by liquid scintillation counting. We assumed that specific activities of the synthesized C16-[14C]ceramide and its isomers were identical to the specific activity (55 mCi/mmol) of the precursor [14C]palmitic acid.Preparation of d-erythro-[3H]Ceramides Having Different Acyl Chain Lengths—d-erythro-[3H]Ceramides (20 mCi/mmol) having different acyl chain lengths (C14–C24) were synthesized by using d-erythro-[3H]sphingosine and various fatty acids through the fatty acid transfer reaction catalyzed by SCDase. Briefly, 174 pmol of d-erythro-[3-3H]sphingosine (23 Ci/mmol), 200 nmol of nonradioactive d-erythro-sphingosine, and 240 nmol of various fatty acids (C14-, C16-, C18-, C20-, C22-, or C24-saturated fatty acid or C24:1-monounsaturated fatty acid) were mixed and incubated in 1 ml of buffer A containing 0.5 milliunits of SCDase at 37 °C for ∼24 h. Other procedures were essentially the same as those for the preparation of [14C]ceramide described above.Preparation of [14C]Dipalmitoylglycerol—[14C]Dipalmitoylglycerol was prepared by phospholipase C treatment of [14C]dipalmitoyl-Ptd-Cho. For the preparation of [14C]dipalmitoylglycerol (10 mCi/mmol), 450 nmol of nonradioactive dipalmitoyl-PtdCho and 50 nmol of l-α-[dipalmitoyl-1-14C]dipalmitoyl-PtdCho (100 mCi/mmol) in chloroform were mixed in a polypropylene tube and dried under a nitrogen gas stream. The dried lipids were suspended in 90 μl of 28 mm Tris-HCl (pH 7.4) buffer by sonication with a probe-type sonicator for 5 min. The suspended lipids were incubated in 150 μl of 16 mm Tris-HCl (pH 7.4) buffer containing 5 mm CaCl2, 0.125% sodium deoxycholate, and 1 unit of C. perfringens phospholipase C at 37 °C for 4 h. The [14C]dipalmitoylglycerol produced was then purified by TLC as described under "Preparation of C16-d-Erythro Types of [14C]Ceramide, [14C]Dihydroceramide, and [14C]Phytoceramide and Unnatural Stereoisomers of C16-[14C]Ceramide by Fatty Acid Transfer Reactions with SCDase," except that a solvent system of hexane/diethylether/acetic acid (70:30:1, v/v) was used for TLC of [14C]dipalmitoylglycerol.Purification of Recombinant CERT and CERTΔST—His6-tagged recombinant human CERT and its START domain-deleted mutant CERTΔST were purified as described previously (8Hanada K. Kumagai K. Yasuda S. Miura Y. Kawano M. Fukasawa M. Nishijima M. Nature. 2003; 426: 803-809Crossref PubMed Scopus (815) Google Scholar, 36Dowler S. Currie R.A. Campbell D.G. Deak M. Kular G. Downes C.P. Alessi D.R. Biochem. J. 2000; 351: 19-31Crossref PubMed Scopus (473) Google Scholar).Intermembrane Ceramide Transfer Assay—Intermembrane transfer of ceramide and its relatives was assayed in a cell-free system that we briefly described previously (8Hanada K. Kumagai K. Yasuda S. Miura Y. Kawano M. Fukasawa M. Nishijima M. Nature. 2003; 426: 803-809Crossref PubMed Scopus (815) Google Scholar). Here we describe the method in more detail.On the day of the lipid transfer assay, purified recombinant CERT and CERTΔST in 10 mm Tris-HCl buffer (pH 7.4) containing 250 mm sucrose buffer were diluted to 1 nmol/ml (71.8 and 45.7 μg/ml for the His6-tagged CERT and CERTΔST, respectively) with buffer C (20 mm Hepes-Na buffer (pH 7.4) containing 50 mm NaCl, and 1 mm EDTA). Donor vesicles per assay consist of 32 nmol of egg PtdCho, 8 nmol of egg PtdEtn, 4 nmol of porcine lactosylceramide, and 0.5 nmol of radioactive ceramide (27.5 nCi for [14C]ceramides or 10 nCi for [3H]ceramides). Acceptor vesicles per assay consist of 320 nmol of egg PtdCho and 80 nmol of egg PtdEtn. Note that the excess amount of acceptor vesicles to donor vesicles is crucial to minimize donor-to-donor transfer of ceramide, which interferes with the donor-to-acceptor transfer reaction. When necessary, donor vesicles additionally contain [3H]dipalmitoyl-PtdCho (125 nCi per assay) as a nonexchangeable lipid marker. According to the number of assays, appropriate amounts of lipids dissolved in organic solvents were mixed in a polypropylene tube (Eppendorf) and dried under a nitrogen gas stream. After addition of buffer C, phospholipid vesicles were prepared by sonication with a probe-type sonicator (model UP-50H, Dr. Hielscher GmbH, Teltow, Germany) at 80% output and 50% cycle for 10 min in a water bath at room temperature. The volume of buffer C that should be added at this step was 20 μl per assay in donor vesicles and 60 μl per assay in acceptor vesicles (note that at least 200 μl of the buffer is required for the sonication step). To remove lipid aggregates, the sonicated samples were centrifuged at 20,000 × g for 30 min at 4 °C, and the supernatant fraction was collected as small vesicles. The radioactivity of the supernatant was determined by liquid scintillation counter for assessing the recovery yields after pre-centrifugation. In some cases, the recovery of lipids in the supernatant fraction was also assessed by the lipid phosphorous quantification method (37Rouser G. Siakotos A.N. Fleischer S. Lipids. 1966; 1: 85-86Crossref PubMed Scopus (1313) Google Scholar). Both assessments showed that over 90% of lipids were reproducibly recovered in the supernatant fraction. The prepared small vesicles were used for intermembrane ceramide transfer assay as follows. In typical experiments, 18 μl of buffer C, 60 μl of acceptor vesicles, and 2 μl of recombinant CERT or CERTΔST (1 nmol/ml in buffer C) were mixed in a 1.5-ml polypropylene tube. Then 20 μl of donor vesicles was added to the tube to start the ceramide transfer reaction. After tapping the tube quickly, the reaction mixture was incubated for 10 min at 37 °C. For mock incubation, buffer C as the vehicle buffer was added in place of the recombinant protein. To stop the reaction, 30 μl of R. communis agglutinin (2.5 mg/ml in phosphate-buffered saline) was added to the reaction mixture and mixed by pipetting. The agglutinin selectively aggregates donor vesicles by cross-linking of the terminal galactose residue of lactosylceramide embedded in donor vesicles. The mixture was chilled on ice for 10 min and centrifuged (20,000 × g, 3 min, 4 °C) to precipitate agglutinated donor vesicles. Then 115 μl of the supernatant fluid was carefully retrieved, and the radioactivity of the supernatant was measured in 2 ml of ACS-II® (Amersham Biosciences) by liquid scintillation counting. To remove the radioactivity due to incomplete precipitation of donor vesicles, the radioactivity from the mock incubation without any CERT recombinants was subtracted from the radioactivity of each sample. When the effects of HPA-12 and its derivatives on ceramide transfer activity were examined, several modifications were made. Specifically, in preparation of donor vesicles, the amount of C16-[14C]ceramide added was reduced from 0.5 to 0.1 nmol. Then 913 μl of buffer C, 20 μl of donor vesicles, 1–5 μl of 3 mm drugs and the vehicle dimethyl sulfoxide (the final concentration of dimethyl sulfoxide was adjusted to 0.5%), and 2 μl of recombinant CERT or CERTΔST (1 nmol/ml) were mixed in a 1.5-ml tube. After preincubation of the mixture for 5 min at 37 °C, 60 μl of acceptor vesicles was added to the mixture and incubated for 30 min at 37 °C. Then after addition of 30 μl of R. communis agglutinin (2.5 mg/ml), the chilled mixture was centrifuged (20,000 × g, 3 min, 4 °C), and 960 μl of the supernatant fluid was retrieved for liquid scintillation counting.Binding Assay of C5-DMB-Ceramide, C6-NBD-Ceramide, and LPS Alexa Fluor® 488 to CERT—On the day of the assay, frozen stocks of recombinant CERT and CERTΔST dissolved in 10 mm Tris-HCl (pH 7.4) containing 150 mm NaCl (Tris-buffered saline; TBS) were thawed and centrifuged (20,000 × g, 4 °C, 10 min) to remove aggregates. The supernatant fraction was retrieved, and its protein concentration was determined. For each binding assay, 400 pmol of His6-tagged CERT or CERTΔST (28.7 or 18.3 μg, respectively) in 118 μl of TBS was mixed in a polypropylene tube (Eppendorf). For a negative control, TBS was added in place of the recombinant proteins. After adding 2 μl of 0.1 mm ethanolic stock solution of C5-DMB-ceramide or C6-NBD-ceramide and 60 μl of TBS to the tube, the mixture was incubated at 37 °C for 30 min for the binding reaction. Then 60 μl of 50% (v/v) slurry of TALON metal affinity resin pre-equilibrated with buffer B was added to the binding reaction mixture and incubated for 10 min at room temperature with rotary shaking. After centrifugation (20,000 × g, 10 s), the supernatant was retrieved as the "unbound fraction." For washing, the resin was suspended in 150 μl of buffer B containing 10 mm imidazole and precipitated, and the supernatant was retrieved as the "wash fraction." This washing step was repeated. The TALON-bound protein was then eluted by incubation with 150 μl of buffer B containing 250 mm imidazole for 5 min at room temperature with occasional tapping. After centrifugation (20,000 × g, 10 s), the supernatant was retrieved as the "elute fraction." A 3.75-fold volume of chloroform/methanol (1:2, v/v) was then added to each retrieved fraction, mixed, and centrifuged (20,000 × g, 10 s). In addition, to retrieve fluorophores that were nonspecifically bound to the resin and tube, 170 μl of TBS and 750 μlof chloroform/methanol (1:2, v/v) were added to the tube containing the resin used, mixed, and centrifuged (20,000 × g, 10 s). The supernatant was retrieved as the "residual fraction." The DMB (excitation at 480 nm; emission at 515 nm) and NBD (excitation at 470 nm; emission at 530 nm) fluorophores in these fractions were quantified with a fluorescence spectrophotometer (model F-3000, Hitachi, Tokyo, Japan). When the binding stoichiometry of CERT and ceramide was analyzed, some modifications were made, because the amount of ceramide must be in excess to that of CERT for this analysis. Briefly, various concentrations of C5-DMB-ceramide were mixed with 40 pmol of recombinant CERT or CERTΔST and 15 μl of 50% slurry of TALON metal affinity resin, and then the mixture (the volume of which was 135 μl) was incubated at 37 °C for 30 min. The amounts of the recombinant proteins distributed to the elute fraction were estimated by densitometric analysis after a portion of the fraction was subjected to SDS-PAGE and Coomassie Blue® staining, using calibration patterns made with known amounts of CERT and CERTΔST. The amount of C5-DMB-ceramide in the elute fraction was quantified as described above.Binding of LPS Alexa Fluor® 488 to recombinant proteins was assayed by essentially the same procedures except that the fluorescent intensity of LPS Alexa Fluor® 488 (excitation at 488 nm; emission at 538 nm) distributed to each fraction was measured without organic solvent extraction.Effect of (1R,3R)-HPA-12 on Metabolic Labeling of Lipids with [14C]Serine in CHO Cells—LY-A, a CHO-K1-derived mutant cell line, is defective in the trafficking of ceramide from the endoplasmic reticulum to the Golgi apparatus because of
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