Phospholipids Into Mammalian Cells Antitumor and Bioactive Choline

The Journal of Immunology

Human TMEM30a Promotes Uptake of Antitumor and Bioactive Choline Phospholipids into Mammalian Cells

Rui Chen, Erin Brady, and Thomas M. McIntyre

Antitumor alkylphospholipids initiate apoptosis in transformed HL-60 and Jurkat cells while sparing their progenitors. 1-O-Alkyl- 2-carboxymethyl-sn-glycero-3-phosphocholine (Edelfosine) like other short-chained phospholipids—inflammatory platelet- activating factor (PAF) and apoptotic oxidatively truncated phospholipids—are proposed to have intracellular sites of action, yet a conduit for these choline phospholipids into mammalian cells is undefined. Edelfosine is also accumulated by Saccharomyces cerevisiae in a process requiring the membrane protein Lem3p, and the human genome contains a Lem3p homolog TMEM30a. We show that import of choline phospholipids into S. cerevisiae DLem3 is partially reconstituted by human TMEM30a and by Lem3p-TMEM30a chimeras, showing the proteins are orthologous. TMEM30a–GFP chimeras expressed in mammalian cells localized in plasma membranes, as well as internal organelles, and ectopic TMEM30a expression promoted uptake of exogenous choline and ethanolamine phospholipids. Short hairpin RNA knockdown of TMEM30a reduced fluorescent choline phospholipid and [3H]PAF import. This knockdown also reduced mitochondrial depolarization from exogenous Edelfosine or the mitotoxic oxidatively truncated phospholipid azelaoyl phosphatidylcholine, and the knockdown reduced apoptosis in response to these two phospholipids. These results show that extracellular choline phospholipids with short sn-2 residues can have intracellular roles and sites of metabolism because they are transport substrates for a TMEM30a phospholipid import system. Variation in this mechanism could limit sensitivity to short chain choline phospholipids such as Edelfosine, PAF, and proapoptotic phospholipids. The Journal of Immunology, 2011, 186: 3215–3225.

-O-Alkyl-2-carboxymethyl-sn-glycero-3-phosphocholine ponent, Lem3p (16) or Ros3p (17), for this internalization. Lem3p (Edelfosine) is a proapoptotic agent with selectivity toward aids import of Edelfosine, fluorescent phosphatidylcholine (PC) 1 tumor cells (1) including promyelocytic leukemic HL-60 (16), fluorescent phosphatidylethanolamine (17), and fluorescent cells (2) and immortalized lymphocytic Jurkat cells (3). Its mode lysophosphatidylethanolamine (18) but not fluorescently labeled of action is yet to be firmly established, but all of its effects from phosphatidylserine. There may be additional transport conduits peroxide-dependent mitochondrial depolarization and apoptosis because varying the fluorescent group on PCs determines whether (2–5) to inhibition of CTP:choline phosphate cytidylyltransferase import of the lipid is aided by Lem3p (19). Humans express two

by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. (6) activity to disruption of lipid raft domains (7–10) all require its RNAs, TMEM30a and TMEM30b, with open reading frames that internalization. However, in contrast to well documented phos- encode proteins of unknown function whose sequences are highly pholipid export proteins (11–14), evidence for phospholipid im- homologous to that of yeast Lem3p (12, 20). port or the proteins that might accomplish this task in mammalian Platelet-activating factor (PAF) (1-O-alkyl-2-acetyl-sn-glycero- cells is meager. Indeed, the best characterized example ATP8B1, 3-phosphocholine) is an intercellular agonist that activates all cells homologous to yeast phospholipid import ATPases, has now been of the innate immune system, and a number of other cells, through found to neither import phosphatidylserine nor affect its asym- a single G protein-coupled PAF receptor expressed on the plasma metric distribution across membrane bilayers (15). membrane of responding cells (21–23). However, the PAF re- The uncertainty of the molecular mechanism of phospholipid ceptor is also enriched in an endosomal compartment (24) and http://classic.jimmunol.org import in mammalian cells stands in contrast to our understand- at the nuclear membrane (25), and nuclear PAF receptors have ing of phospholipid uptake by yeast. Saccharomyces cerevisiae unassailably been shown to initiate nuclear Ca2+ ﬂux and gene accumulate phospholipids from their external environment, and transcription (26, 27)—illuminating a potential intracellular role two independent approaches, one of which was selection for re- for PAF. The PAF receptor has a critical role in the metastatic sistance to Edelfosine toxicity, identiﬁed the same essential com- distribution of melanoma cells and is a potential therapeutic tar- get (28). Whether the nuclear PAF receptor responds to PAF pro-

Downloaded from duced by distal cells or solely responds to intracellular PAF pro- Department of Cell Biology, Cleveland Clinic, Cleveland, OH 44195 duction is unknown because PAF is not known to readily transit Received for publication August 10, 2010. Accepted for publication December 17, 2010. mammalian plasma membranes (29). PAF is, however, readily in- This work was supported by Grants P01 HL087018, HL092747, AA017748, and P50 ternalized by S. cerevisiae when given the opportunity (30). HL081011 from the National Institutes of Health. An oxidative attack on polyunsaturated phospholipids generates Address correspondence and reprint requests to Dr. Thomas M. McIntyre, Depart- a host of products, some of which are short chain phospholipids ment of Cell Biology, NE10, Lerner Research Institute, Cleveland Clinic Lerner created by oxidative scission of the sn-2 polyunsaturated fatty acyl College of Medicine, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: [email protected] residue. Oxidatively truncated phospholipids, like the abundant Abbreviations used in this article: Az, azelaoyl; CHO, Chinese hamster ovary; NBD, oxidation product azelaoyl (Az)-PC, induce apoptosis through nitrobenzoxadiazole; OFP, orange ﬂuorescent protein; PAF, platelet-activating factor; a direct effect on mitochondria that initiates the intrinsic caspase PC, phosphatidylcholine; shRNA, short hairpin RNA; TMEM30a, transmembrane 30 cascade (31, 32). The rapid incorporation of exogenous Az-PC kDa a; TMEM30b, transmembrane 30 kDa b. into intracellular mitochondrial membranes (32) suggests the pres- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 ence of a cellular uptake mechanism.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002710 3216 TMEM30a IN PHOSPHOLIPID IMPORT

We find (33) that intravascular PAF or oxidatively truncated were confirmed by sequencing and Western blotting when appropriate. phospholipids are rapidly (t ∼ 30 s) cleared from the circulation The TMEM30a-GFP construct PCR product of human TMEM30a with 1/2 9 and that the mechanism is not primarily intravascular hydrolysis 3 -primer GGCGACCGGTGGAATGGTAATGTCAGCTGTATTAC and 59-primer GGGGGAATTCCTGTCAGGGTCAGCCGGC was digested by by plasma PAF acetylhydrolase (34) as expected. Instead, we find EcoRI and AGeI and inserted into pd2EGFP-N1 vector. that these short chain phospholipids are rapidly internalized as intact phospholipids by an undefined, saturable mechanism. This TMTM30a silencing suggests that import of short-chained phospholipids has a critical shRNA against human TMEM30a with insert sequences 59-GGATGA- role in mammalian cells in vivo. TCTTGAGCACTATTT-39,59-CCAGTTTACATTGCTGGATTCT-39,59- 9 9 In this article, we show that TMEM30a is a functional ortholog TGTGAACCTTATCGAAGAAT-3 ,5-CAGTCCCTGTAATAAATGTTT- 39, or control sequence 59-CAGTCCTGTAATAAATGTTT-39 were sepa- of S. cerevisiae Lem3p that aids internalization of the antitumor rately transfected into Jurkat cells by nucleofection before selection in phospholipid Edelfosine, the short chain inflammatory lipid PAF, 500 nM neomycin. and the mitotoxic phospholipid Az-PC in yeast and in mammalian TMEM30a expression cells. Thus, short chain choline phospholipids found in the extracellular environment during atherogensis (35), chronic ethanol Overnight cultures of S. cerevisiae were diluted and grown to confluence ingestion (36), UV irradiation (37), or acute cigarette smoke ex- in 2% raffinose, recovered by centrifugation, and then grown in uracil posure (38) can be become intracellular effectors. dropout media with glucose or 0.4% galactose containing 2% raffinose such that they grew for ∼4 h to log phase (OD600 0.4–0.6). The cells were collected by centrifugation (3000 3 g, 5 min, room temperature) and Materials and Methods washed twice with sterile Millipore water before being resuspended in CelLytic Cell Lysis reagent. Glass beads (0.5 mm) were added (10% w/v), Materials and cells were lysed by shaking twice at 4800 rpm for 30 s in a mini- 3 Chinese hamster ovary (CHO) K1 cells and S. cerevisiae stains 201388 beadbeater. The resulting lysates were centrifuged (12,000 g, 10 min, and 4001121 were from American Type Culture Collection (Manassas, 4˚C) before the proteins resolved by electrophoresis in a 10% SDS poly- VA). Oligonucleotide primers, MitoTracker Orange CMTMRRos, Organelle acrylamide gel. Anti-V5 (1:2500) and HRP-conjugated goat anti-mouse Lights Golgi-orange fluorescent protein (OFP), Organelle lights ER-OFP, Ab (1:5000) were used to detect the expression of V5-tagged Lem3, pYES2/CT vector, anti-V5 Ab, FBS, and S. cerevisiae EasyComp Trans- hTMEM30a, or chimeric proteins in S. cerevisiae. Human HepG2 cells formation kit, were products from Invitrogen Life Technologies. Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and short hairpin RNA (shRNA) plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD). RNeasy Mini kits were from Qiagen (Valencia, CA). Az-PC, nitrobenzoxadiazole (NBD)- PC, NBD-PE, Edelfosine, and PAF were obtained from Avanti Polar Lipid (Alabaster, AL). [2-[3H]acetyl]PAF was a product of PerkinElmer Life and Analytical Sciences (Boston, MA). Anti-GFP was from Santa Cruz Biotechnology, and mounting medium was a product of Vector Laboratories (Burlingame, CA). Anti-Na/K ATPase was from Abcam (Cambridge, MA). DMEM, Ham’s F-12, RPMI 1640 medium, and penicillin/streptomycin were obtained from the Cleveland Clinic media core, whereas yeast nitrogen base with amino acids, synthetic dropout by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. media supplement without uracil, CelLytic Cell Lysis reagent, and other reagents were obtained from Sigma-Aldrich. Cell culture CHO, HepG2, and Jurkat cells were cultured in DMEM:F-12 and RPMI 1640 medium, respectively, with 10% FBS at 37˚C in an atmosphere containing 5% CO2. S. cerevisiae strains 4001121 or 201388 were cultured in YPD medium with rotation at 270 rpm at 28˚C. Transformants were cultured in uracil dropout media with the stated carbon source. Cell number was assessed either by serial 2-fold dilutions, followed by plating http://classic.jimmunol.org on appropriate agar media or in liquid culture by scattering at OD600. Expression constructs The sequences used to amplify human TMEM30a or S. cerevisiae Lem3 to generate expression constructs or yeast/human chimeras are as follows: Lem3, 59-GTGCTGGAATTCCATCAGTACAGACCAGAAAA, and 39-G- GCTCTAGATTTCATATTCCATGACAAAC; hTMEM30a, 59-GAATAT-

Downloaded from TAAGCTTACCATGGTAAATAACTATAACGCGAAGGATGAAG, and 39-GGCTCTAGAAATGGTAATGTCAGCTGTATTAC; Lem3/hTMEM30- a chimera (LT), 59-TAATACGACTCACTATAGGG, and 39-CTATCATCT- CGAGATAGTACATATCTTCTGTG; TMEM30a/Lem3 chimera (TL), 59- CTGGGGATCCATTTGTGCTGCCATGGCATTG, and 39-GCGTGAAT- GTAAGCGTGAC; and TMEM30a/Lem3/TMEM30a chimera (TLT), 59- GGGCTCGAGATTTTAGCGAAGATCAGATA, and 39-GGAGATGGAT- CCACAGCCTCCAATCAGGT. Lem3 sequence was generated by RT-PCR using 201388 yeast strain RNA with SuperScrip III Reverse Transcriptase and Vent polymerase with the stated Lem3 primers. This PCR product was inserted into pYES2/CT by EcoRI and XbaI digestion, ligated, and transformed into DH5a. The hTMEM30a PCR product was digested with HindIII FIGURE 1. Lem3 promotes uptake of short-chained PCs by S. cer- and XbaI and similarly introduced into pYES2/CT. The Lem3 and evisiae A TMEM30a chimeric constructs were made starting with the TMEM30a . , Flow cytometric analysis of NBD-PC uptake by wild-type or D pYES2/CT plasmid and the primers listed above to produce appropriate Lem3 S. cerevisiae. B, Edelfosine concentration-dependent suppression Lem3 fragments. The Lem3 fragments and hTMEM30a-Yes2/CT were of wild-type or DLem3 growth. C, Competition for NBD-PC uptake by digested by the stated restriction enzymes and the resulting fragments ligated wild-type S. cerevisiae by an excess of the cytotoxic choline phospholipid and transformed, according to the manufacturer’s directions. All constructs Edelfosine or the oxidatively truncated PC Az-PC. The Journal of Immunology 3217

were lysed by radioimmunoprecipitation assay buffer and centrifuged were verified by Western blot analysis. The cells were seeded on coverslips (12,000 3 g, 25 min, 4˚C) before these supernatants were resolved by 10% in 6-well plates 1 d prior to staining. For organelle labeling, organelle SDS-PAGE. Expressed proteins were detected using murine anti-GFP lights Golgi-OFP and organelle lights ER-OFP were used to label Golgi (1:1000) and plasma membrane human (because Chinese hamster is not body and endoplasmic reticulum, respectively, following the manu- commercially available) anti-NaK ATPase HRP-conjugated goat anti- facturer’s procedure. The culture medium was aspirated, and 550 ml or- mouse (1:5000). ganelle lights transduction solution was added to the well before incubation for 4 h at room temperature. After the solution was removed, Lipid accumulation serum-free DMEM:F-12 medium containing enhancer was added to the Jurkat cells, transfected with TMEM30a shRNA or not, were washed with well and incubated for 2 h at 37˚C in a 5% CO2 atmosphere. The cells were 6 washed in HBSS before fixation in 3.75% formaldehyde for 10 min, fol- HBSS twice and resuspended (2 3 10 cells/ml) in 1 mM NDB-PC or lowed by washing three times with PBS. The coverslip containing the cells NDB-PE and kept at room temperature for 10 min. Competition for NBD- PC (1 mM) uptake by TMTM30a-Jurkat cells used 5 mM unlabeled was mounted with DAPI containing media prior to fluorescence imaging. For plasma membrane staining, the cells were washed with serum-free phospholipid and the same conditions. The labeled cells were washed m twice with HBSS containing 1% (w/v) BSA before analysis by flow medium once, and medium containing 2.5 g/ml CellMask orange cytometry. To assess [3H]PAF uptake, transfected CHO cells were seeded plasma membrane stain solution was added for a 5-min incubation at 37˚C in 12-well plates (6 3 105 cells/well) 1 d before labeling. These were then before the cells were processed as above. Mitochondria were labeled by 3 washing the cells three times with HBSS before 25 nM MitoTracker or- washed with PBS three times before incubation with 75,000 dpm [2-[ H] ange CMTMRRos in HBSS was added to each well. The cells were in- acetyl]PAF in 500 ml HBSS containing 0.005% BSA with or without 1 mM unlabeled PAF for 10 min either at room temperature or on ice. The cells cubated at 37˚C for 15 min, washed three times with PBS, mounted, and were then washed twice with cold 1% BSA in PBS and then once with immediately imaged. All confocal fluorescent micrographs are an image of xy PBS. The cells were detached with trypsin/EDTA and transferred to a single plane. Ecolite (CPM Biomedicals, Solon, OH) scintillation fluid for quantitation. Annexin V labeling Lipid uptake by S. cerevisiae was determined using sn-1 NBD-PC, NBD-phosphatidylethanolamine, or NBD-phosphatidylserine. S. cerevi- TMEM30a-GFP–transfected CHO cells or control cells were detached siae was grown overnight in uracil dropout media with raffinose as a car- nonenzymatically with Cellstripper (Mediatech, Herndon, VA), washed bon source. The following morning the cells were diluted to 0.2 OD600 in with complete medium once, and then with annexin V binding buffer [10 medium containing glucose or galactose. The cells were cultured for 6 h mM HEPES/NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2] before and washed with PBS three times before incubation with 1 mM NBD- being suspended (3 3 106 cells/ 100 ml) in this buffer with or without 5 ml labeled phospholipid in PBS for 10 min at room temperature. The la- annexin V-Alexa647 conjugate (Invitrogen) and kept at room temperature beled cells were washed with PBS containing 1% BSA three times and for 15 min. The cells were then diluted with 1 ml binding buffer for flow suspended in the same buffer for flow cytometry. cytometry. Colony formation on phospholipid agar plates was tested by growing S. cerevisiae in glucose dropout media overnight before dilution to 0.2 OD600 Cell fractionation in raffinose dropout media. After growth in log phase to 0.4–0.6 OD , the 8 600 TMEM30a-GFP stable transfectants (2 3 10 ) were washed three times cells were diluted to 0.01 OD in raffinose dropout media. This was serially m with PBS and resuspended in 5 ml extraction buffer [0.25 M sucrose, diluted 2-fold, and 3 l of each dilution was spotted on an agar plate 10 mM HEPES (pH 7.4), 1 mM EDTA, and protease inhibitor mixture containing the stated reagent before growth in a 30˚C humid atmosphere. (Roche)] before being equilibrated at 500 psi N2 for 15 min in a nitrogen Organelle labeling cavitation bomb (Parr Instrument, Moline, IL). After explosive de- pressurization, the resulting lysate was centrifuged (500 3 g, 5 min, 4˚C) CHO and HepG2 cells were stably transfected with a human TMEM30a- to remove cell debris before being layered over a 0.6–2 M continuous GFP chimeric expression plasmid and grown in 500 mg/ml G418 before sucrose gradient for density separation (28,000 rpm, SW41 rotor) for 90 by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. single clones were selected by sorting for GFP. Highly expressing clones min at 4˚C without braking. Aliquots of the fractions collected from the

Table I. Sequence alignment of S. cerevisiae Lem3p, human TMEM30a, and their chimeras

Lem3 MVNFDLGQVGEVFRRKDKGAIVSGDNPEEEEDVDASEFEEDEVKPVRTKNRRPKEDAFTQQRLAAINPVLTPRTVLPLYLLIAVVFVI TMEM30a ------MAMNYNAKDEVDGGPPCAPGG-----TAKTRRPDNTAFKQQRLPAWQPILTAGTVLPIFFIIGLIFIP LT MVNFDLGQVGEVFRRKDKGAIVSGDNPEEEEDVDASEFEEDEVKPVRTKNRRPKEDAFTQQRLAAINPVLTPRTVLPLYLLIAVVFVI TL MVNFDLGQVGEVFRRKDKGAIVSGDNPEEEEDVDASEFEEDEVKPVRTKNRRPKEDAFTQQRLAAINPVLTPRTVLPLYLLIAVVFVI TLT ------MAMNYNAKDEVDGGPPCAPGG-----TAKTRRPDNTAFKQQRLPAWQPILTAGTVLPIFFIIGLIFIP http://classic.jimmunol.org Lem3 VGGCILAQNSKVDEVTIYYQDCMTNATSSWSDIPSEHWQFVFHKYKTYNTAPQWRFVDDESDDFTKQRGTCQIRFTTPSDMKNNVYLN TMEM30a IGIGIFVTSNNIREIEIDYTGTEPSSP------CNKCLSPDVTPCF------CTINFTLEKSFEGNVFMY LT VGGCILAQNSKVDEVTIYYQDCMTNATSSWSDIPSEHWQFVFHKYKTYNTAPQWRFVDDESDDFTKQRGTCQIRFTTPSDMKNNVYLN TL VGGCILAQNSKVDEVTIYYQDCMTNATSSWSDIPSEHWQFVFHKYKTYNTAPQWRFVDDESDDFTKQRGTCQIRFTTPSDMKNNVYLN TLT IGIGIFVTSNNIREIEIDYTGTEPSSP------CNKCLSPDVTPCF–------CTINFTLEKSFEGNVFMY

Lem3 YVLEKFAANHRRYVLSFSEDQIRGEDASYETVHDATGINCKPLSKNADGKIYYPCGLIANSMFNDTFPLQLTNVGDTSNNYSLTNKGI TMEM30a YGLSNFYQNHRRYVKSRDDSQLNGDSSALLNPSKE----CEPYRRNED-KPIAPCGAIANSMFNDTLELFLIGNDSYPIPIALKKKGI Downloaded from LT YVLEKFAANHRRYVLSFSDSQLNGDSSALLNPSKE----CEPYRRNED-KPIAPCGAIANSMFNDTLELFLIGNDSYPIPIALKKKGI TL YVLEKFAANHRRYVLSFSDSQLNGDSSALLNPSKE----CEPYRRNED-KPIAPCGAIANSMFNDTLELFLIGNDSYPIPIALKKKGI TLT YGLSNFYQNHRRYVLSFSEDQIRGEDASYETVHDATGINCKPLSKNADGKIYYPCGLIANSMFNDTFPLQLTNVGDTSNNYSLTNKGI

Lem3 NWESDKKRYKKTKYN------YTQIAPPPYWEKMYPDGYNETNIPDIQDWEEFQNWMRPGAFDKITKLIRINK-----NDTLPAGEY TMEM30a AWWTDKNVKFRNPPGGDNLEERFKGTTKPVNWLKPVYMLDSDPDNNGFIN-EDFIVWMRTAALPTFRKLYRLIERKSDLHPTLPAGRY LT AWWTDKNVKFRNPPGGDNLEERFKGTTKPVNWLKPVYMLDSDPDNNGFIN-EDFIVWMRTAALPTFRKLYRLIERKSDLHPTLPAGRY TL AWWTDKNVKFRNPPGGDNLEERFKGTTKPVNWLKPVYMLDSDPDNNGFIN-EDFIVWMRTAALPTFRKLYRLIERKSDLHPTLPAGRY TLT NWESDKKRYKKTKYN------YTQIAPPPYWEKMYPDGYNETNIPDIQDWEEFQNWMRPGAFDKITKLIRINK-----NDTLPAGEY

Lem3 QLDIGLHWPVLEFNGKKGIYLTHGSHLGGRNPFLGIVYLIGGCICAAMALILLTFWLFGGRKIADASSLSWNMK TMEM30a SLNVTYNYPVHYFDGRKRMILSTISWMGGKNPFLGIAYIAVGSISFLLGVVLLVIN-HKYRNSSNTADITI--- LT SLNVTYNYPVHYFDGRKRMILSTISWMGGKNPFLGIAYIAVGSISFLLGVVLLVIN-HKYRNSSNTADITI--- TL SLNVTYNYPVHYFDGRKRMILSTISWMGGKNPFLGIAYIAVGCICAAMALILLTFWLFGGRKIADASSLSWNMK TLT QLDIGLHWPVLEFNGKKGIYLTHGSHLGGRNPFLGIVYLIGGCISFLLGVVLLVIN-HKYRNSSNTADITI--- S. cerevisiae Lem3p (bold) sequences aligned with human TMEM30a sequences, with dashes denoting breaks. Underlined Lem3p sequences deﬁne the two predicted transmembrane sequences. Chimeras formed from Lem3p (L) and TMEM30a (T) are also aligned to the S. cerevisiae protein. 3218 TMEM30a IN PHOSPHOLIPID IMPORT

bottom of the gradient were mixed with sample buffer for protein reso- lution by SDS-PAGE, followed by Western blotting for GFP and plasma membrane markers.

Results Lem3p enhances short chain phospholipid import in S. cerevisiae Wild-type S. cerevisiae accumulate NBD-labeled phospholipids, which have relatively high critical micellar concentrations and are soluble, from their environment (Fig. 1A). This uptake was dependent on Lem3p because loss of this membrane protein greatly reduced this influx. The phospholipid Edelfosine, with its very short methyl ether sn-2 residue, also is imported by S. cerevisiae where—as with transformed mammalian cells—it induced cell death (Fig. 1B). Cells in which Lem3 was genetically ablated were resistant to this cytotoxic phospholipid. Short-chained phospholipids, including the truncated phospholipid oxidation product Az-PC, induce cell death through a direct physical interaction with mitochondria (31, 32)—a process that necessarily includes internalization of the exogenous lipid. We tested whether this phospholipid with a 9-carbon long sn-2 residue was also a Lem3p transport system substrate by examining its ability to compete for uptake of the fluorescent phospholipid NBD-PC. We found that excess Edelfosine effectively reduced uptake of the fluorescently labeled phospholipid (Fig. 1C) and that Az-PC similarly reduced, although less effectively, uptake of the labeled transport substrate.

Human TMEM30a partially reconstitutes phospholipid import in DLem3 S. cerevisiae The human genome contains two sequences, transmembrane 30 kDa FIGURE 2. Human TMEM30a partially reconstitutes PC uptake in S. cerevisiae lacking Lem3p. A, DLem3 S. cerevisiae transformed with empty a and b (TMEM30a and TMEM30b). These sequences would encode vector or two isolates transformed with human TMEM30a were grown on proteins that are 39% identical with 54% similar residues to S. galactose to induce TMEM30a expression. NBD-PC uptake was de- cerevisiae Lem3p (Table I). The human sequence also retains the termined by flow cytometry. B, Concentration-dependent effect of Edel- highly conserved sequence QNHRRYVKS found in nearly all of by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. fosine on colony growth of serially diluted wild-type S. cerevisiae or this family’s homologs. We expressed human TMEM30a in the DLem3 transformed with empty vector or two DLem3 isolates transformed DLem3 mutant under the control of a Gal-1 promoter. This trans- with human TMEM30a. gene had no effect on NBD-PC accumulation in S. cerevisiae grown on glucose where the protein was not expressed but did promote the with Lem3p, although expression of the chimeras or full-length uptake of the fluorescent phospholipid in either of two isolates TMEM30a was always significantly less than when Lem3p was when grown on galactose to induce the protein (Fig. 2A). reintroduced in DLem3 (Fig. 3B). We found that DLem3 trans- S. cerevisiae is sensitive to the cytotoxic drug Edelfosine, so we formed with Lem3p effectively imported NBD-PC, that human determined whether human TMEM30a would increase the sensi- TMEM30a modestly enhanced lipid uptake in the DLem3 mutant, D http://classic.jimmunol.org tivity of the resistant Lem3 mutant to its toxic effect. Serial di- and that an N-terminal Lem3p/C-terminal TMEM30a chimera lution of wild-type S. cerevisiae transformed with empty vector, (LT; Table I) was more effective than the human sequence in m followed by culture on galactose plates, showed that 10 g/ml promoting fluorescent phospholipid accumulation (Fig. 3A,3B). Edelfosine reduced viability and that loss of cell viability was The mirror image chimera (TL) with an N-terminal TMEM30a m more pronounced when the cells were plated on 20 g/ml Edel- and C-terminal Lem3p was equally effective in promoting uptake D fosine (Fig. 2B). The Lem3 mutant transformed either with of NBD-PC (Fig. 3B). A chimera (TLT) where only the central empty vector or the TMEM30a vector and grown on repressive Downloaded from portion of the human–yeast–human protein was encoded by m glucose was resistant to the cytotoxic effect of even 20 g/ml Lem3p was less effective than either the yeast–human or human– Edelfosine. The outcomes differed when the cells were grown yeast protein, although its low level of expression compromised its D on galactose to induce TMEM30a. In this case, Lem3 trans- effectiveness. For all of these chimeras, the uptake of NBC-PC formed with empty vector remained resistant to Edelfosine, was greater when the strains were grown on galactose than on whereas expression of either of two TMEM30a clones sensitized glucose where plasmid proteins are not expressed. m these cells to the effect of 10 and 20 g/ml Edelfosine. We determined whether human/yeast chimeras increased the sensitivity of DLem3 to Edelfosine cytotoxicity. As before, the TMEM30a is orthologous to S. cerevisiae Lem3p wild-type parent was sensitive to Edelfosine and the DLem3 mu- We sought to determine whether human TMEM30a is truly tant insensitive when grown on either glucose or galactose (Fig. orthologous to Lem3p by forming chimeras of the two proteins 3C). The DLem3 mutant containing TMEM30a under the control by substituting regions defined by the putative transmembrane of the Gal-1 promoter behaved just as its empty vector in the domains (Table I) to determine whether human encoded regions DLem3 control on glucose plates but became sensitive to Edel- functionally substitute for the yeast sequences they replaced. Yeast fosine when grown on galactose plates when the TMEM30a was expressed human TMEM30a, and all of the chimeras formed expressed. Although not as fully effective as Gal-1–induced The Journal of Immunology 3219 by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. http://classic.jimmunol.org

FIGURE 3. Human TMEM30a-S. cerevisiae Lem3p chimeras partially reconstitute ﬂuorescent PC uptake by S. cerevisiae lacking Lem3p. A, NBD-PC uptake determined by ﬂow cytometry for wild-type S. cerevisiae transformed with empty vector or DLem3 transformed with Lem3, TMEM30a, or a chimera (Table I) of Lem3 and TMEM30a. B, Quantitation (n = 3) of NBD-PC uptake by DLem3 transformed with Lem3-TMEM30a (LT; see Table I for sequence),

Downloaded from TMEM30a-Lem3 (TL), or TMEM30a-Lem3-TMEM30a (TLT) chimeras. Western blot (top panel) for V5 Ag contained in sequences encoding TMEM30a and its chimeras isolated from protein extracts of S. cerevisiae grown in galactose to induce insert expression or noninducing glucose. C, Concentration-dependent effect of Edelfosine on colony formation on glucose or galactose plates for wild-type S. cerevisiae or DLem3 transformed with galactose induced human, yeast, or chimeric constructs. D,EffectofEdelfosineonDLem3 viability after introduction of human TMEM30a, yeast Lem3p, or chimeras formed from them. Cell

number (OD600) in liquid culture of wild-type or DLem3 transformed with the stated vectors at deﬁned concentrations (left panel)or12.5mg/ml (right panel).

Lem3p in reconstituting DLem3’s sensitivity to Edelfosine, the to Edelfosine, as did expression of human TMEM30a, although TMTM30a/Lem3p and Lem3p/TMEM30a/Lem3p chimeras, and the human sequence was signiﬁcantly less effective (Fig. 3D). As especially the TMEM30a/Lem3p chimera, sharply increased the before, each chimera sensitized the DLem3 strain to Edelfosine, sensitivity of DLem3 cells to exogenous Edelfosine. The in- although in this assay all the chimeras were similarly effective. terchangeability of Lem3p and TMEM30a show the proteins are orthologous. TMEM30a-GFP distributes among CHO plasma membrane An identical outcome obtained when the experiment was per- and internal organelles formed in liquid culture where cell numbers could be quantitated. We determined whether human TMEM30a is properly positioned to Reintroduction of Lem3p in the DLem3 strain restored sensitivity import phospholipids into mammalian cells using CHO cells stably 3220 TMEM30a IN PHOSPHOLIPID IMPORT

transfected with TMEM30a-GFP and maintained under G418 se- MitoTracker Red that marks polarized mitochondria. The apparent lection. We observed by confocal microscopy that one population diffuse TMEM30a-GFP staining, then, reflects the wide intracel- of cells abundantly expressed the ectopic protein, with staining lular distribution of the protein. throughout the cytoplasm and a delimiting rim of green fluorescence We confirmed that TMEM30a-GFP associated with internal or- (Fig. 4A). Other cells displayed less intense internal expression of ganelles by separating membranes of HepG2 cells stably trans- the fluorescent chimera, and these cells expressed the TMEM30a- fected with TMEM30a-GFP by sucrose density centrifugation and GFP just as a rim of green fluorescence around the cell. We con- immunoblotting for TMEM30a-GFP and the plasma membrane firmed this rim pattern of fluorescence represented plasma mem- marker Na/K ATPase (Fig. 4B). The TMEM30a chimera was brane localization of TMEM30a-GFP by staining these cells with prominent in the plasma membrane fraction but also was widely the plasma membrane dye CellMask Orange. The rim pattern of distributed among other membrane components. staining of both cells with high expression and those with less TMEM30a aided choline phospholipid uptake by CHO cells abundant TMEM30a-GFP expression produced a rim of orange through a saturable process because excess Az-PC or Edelfosine fluorescence that indicates colocalization at the plasma membrane. decreased fluorescent PC accumulation by TMEM30a-expressing Lem3p-GFP is found in discrete patches in the plasma membrane cells (Fig. 4C). Phosphatidylserine, in contrast, did not compete of S. cerevisiae but also in intracellular structures including the for fluorescent PC uptake. endoplasmic reticulum (17). TMEM30a also was present in CHO cell intracellular structures, and coexpression with endoplasmic TMEM30a enhances phospholipid import into CHO cells reticulum and Golgi Organelle Lights show (Fig. 4A) that the We tested the postulate that TMEM30a participates in phospholipid mammalian protein colocalized with these organelles in cells that import in mammalian cells by examining NBD-PC uptake into strongly expressed TMEM30a-GFP. TMEM30a also localized with CHO cells stably expressing TMEM30a. CHO cells transfected

by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. FIGURE 4. TMEM30a–GFP localizes in the plasma membrane and internal organelles. A, CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask Orange Plasma Mem- brane to mark the plasma membrane (top panels) then imaged by confocal microscopy (original mag- nification 360). Coexpression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum (row 2) or Golgi (row 3). TMEM30a-GFP–expressing CHO cells were labeled http://classic.jimmunol.org with MitoTracker Red to identify polarized mitochondria (bottom panels). B, Western blot for GFP or plasma membrane Na/K ATPase in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. C, Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10-min incubation in the presence of NBD-PC (1 Downloaded from mM) alone or additionally with 5 mM Az-LysoPAF, phosphatidylserine (PS), or Edelfosine. The Journal of Immunology 3221

with empty vector showed that the NBD-PC associated with these alone, has a role in choline phospholipid import that is distinct cells was prominently associated with the cell’s plasma membrane from its role in enabling ATP8B1 uptake of phosphatidylserine. (Fig. 5A, inset). In contrast, cells expressing TMEM30a accumulated more NBD-PC, and a significant portion of this was in- TMEM30a knockdown reduces phospholipid import by Jurkat tracellular and associated with structures that were clearly ex- cells cluded from the nucleus. Edelfosine is selectively cytotoxic for lymphoblastoid Jurkat cells We confirmed that the TMEM30a-GFP chimera that we used compared with naive lymphocytes (4, 9, 40–42), suggesting Jurkat to define the intracellular distribution of TMEM30a retained its cells internalize this short chain phospholipid. We found that function and that TMEM30a participates in the accumulation of the Jurkat cells express mRNA for TMEM30a and that a shRNA di- inflammatory mediator PAF by quantifying the amount of [3H]PAF rected to it suppressed its expression by 70% (Fig. 6A). Jurkat accumulated by transfected CHO cells. We observed that the ex- cells expressing TMEM30a shRNA were less sensitive to the cy- pression of TMEM30a-GFP, or a TMME30a modified by a Lumio totoxic effect of Edelfosine than control cells (Fig. 6B). Jurkat tag that introduces a short sequence that selectively binds a pro- cells accumulated both NBD-PC and NBD-PE as determined by prietary fluorescent dye, enhanced [3H]PAF uptake (Fig. 5B). Thus, flow cytometry (Fig. 6C), and TMEM30a shRNA reduced the the NBD fluorescent group is not required for phospholipid trans- amount of both these phospholipids associated with the cells. The port, and exogenous PAF is a transport substrate of TMEM30a— reduction of phospholipid uptake by the TMEM30a knockdown and so has access to intracellular PAF receptors (24–27). was greater for NBD-PE than NBD-PC (Fig. 6D). We also found TMTM30a expressed by itself does not increase phosphati- that shRNA knockdown of TMEM30a reduced the uptake of dylserine uptake (39), and phosphatidylserine does not compete radiolabeled PAF (Fig. 6E), so exogenous PAF is internalized with for the uptake of choline phospholipids (Fig. 4C). However, when the aid of TMEM30a. TMEM30a is coexpressed with the P4-type ATPase ATP8B1 phosphatidylserine uptake is increased and basal surface expres- TMEM30a knockdown reduces mitochondrial depolarization sion of phosphatidylserine was reduced (39), although this may by exogenous short-chained phospholipids not be a direct ATP81-mediated function (15). We determined Oxidatively truncated phospholipids such as Az-PC are rapidly whether expression of TMEM30a in the absence of coexpressed internalized, migrate to mitochondria (32), and then initiate the APT8B1 reduced the display of phosphatidylserine on the cell mitochondrial-dependent apoptotic cascade (31, 32). We tested the surface. We found that even when TMEM30a-GFP was highly potential role of TMEM30a in internalizing this mitotoxic lipid by abundant that there was no diminution of annexin V staining using shRNA and the potentiometric mitochondrial dye JC-1. This of surface phosphatidylserine (Fig. 5C). This indicates that cationic dye is concentrated, and precipitated, in respiring mito- TMEM30a, either through association with another ATPase or chondria by the electrochemical gradient and so fluoresces red/ by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd.

FIGURE 5. TMEM30a enhances import of http://classic.jimmunol.org short-chained PCs, and not phosphatidylserine, into CHO cells. A, NBD-PC uptake by CHO cells transfected with empty vector or a TMEM30a vector assessed by confocal microscopy (340). Inset, 360. B, Uptake of [3H]PAF by CHO cells expressing TMEM30a containing a GFP or Lumio tag (n = 3). C, Phosphatidylserine surface Downloaded from expression is not reduced in TMEM30a-transfected CHO cells. Surface phosphatidylserine was detected (n = 3) by ﬂow cytometry with

FIGURE 6. shRNA knockdown of Jurkat cell TMEM30a reduces mitotoxicity of exogenous short-chained PCs. A, Quantitative PCR for TMEM30a mRNA after transfection by empty vector or one containing TMEM30a shRNA (n = 3). B, Jurkat viability to Edelfosine exposure after transfection with an empty vector or TMEM30a shRNA (n = 3). C, Jurkat cell uptake of ﬂuorescent NBD-PC (upper panel) or NBD-phosphatidylethanolamine (lower panel)by cells expressing TMEM30a shRNA or its vector (n = 3). D, Quantitation of NBD-PC accumulation by Jurkat cells expressing TMEM30a shRNA or empty vector (n = 3). E, Uptake of [3H]PAF by Jurkat cells is reduced by TMEM30a shRNA knockdown (n = 4). All quantitative measures used triplicate determinations in each experiment.

orange, whereas monomeric dye in the cytoplasm not incorporated 35% of the population. We conclude exogenous Edelfosine and http://classic.jimmunol.org into mitochondria ﬂuoresces in the green channel. We found (Fig. Az-PC both access intracellular mitochondria in part through 7A) that suppression of TMEM30a by shRNA expression reduced TMEM30a-mediated uptake. mitochondrial depolarization caused by Az-PC. Thus, just 57% of the shRNA-transfected cells lost their mitochondrial trans- Discussion membrane potential after exposure to 10 mM Az-PC compared The primary ﬁndings in this work are that mammalian cells readily with the 72% of vector transfected cells that became depolarized internalize exogenous short-chained phospholipids and that they do

Downloaded from by this oxidatively truncated phospholipid. Intracellular mito- so using a transport or flipping system orthologous to the phos- chondria are therefore vulnerable to short-chained phospholipids pholipid import system identified in S. cerevisiae. Indeed, we find in their environment, because these phospholipids are internalized the human protein TMEM30a functions in S. cerevisiae to increase in part through a TMEM30a-dependent process. uptake of exogenous phospholipids in cells whose phospholipid Exogenous Edelfosine also affects mitochondrial function and import system had been crippled by ablating the Lem3p component induces mitochondria-dependent apoptosis (2, 5). We again found of their transport system. The ability of chimeras of the human and just 10% of vector transfected cells were in the lower rightmost bin with low red/orange mitochondrial fluorescence and high yeast protein to also enhance phospholipid import in this genetic green cytoplasmic fluorescence in the absence of Edelfosine (Fig. background shows that the function of this protein has been main- ∼ 7B). This number increased to 70% when the concentration of tained in organisms that diverged 100 million years ago. exogenous Edelfosine was increased to 10 mg/ml, showing that mRNA for TMEM30a is widely expressed, although the pres- Edelfosine effectively depolarized mitochondria. The number of ence of the encoded protein has yet to be reported. We used Jurkat cells transfected with TMEM30a shRNA in the rightmost GFP chimeras to determine where TMEM30a distributed within bin also was 10% of the population in the absence of Edelfo- mammalian cells because an anti-peptide Ab to the conserved sine, but at 10 mg/ml Edelfosine, this number increased to just element we prepared was of too low affinity to be useful in lo- The Journal of Immunology 3223

FIGURE 7. shRNA knockdown of Jurkat cell TMEM30a reduces Az-choline phospholipid and Edelfosine mitotoxicity. A, Flow cytometric analysis of JC-1 green fluorescence (FL1, x-axis) and orange/red fluorescence (FL2, y-axis) in the presence of the stated Az-LysoPAF concentration in vector and TMEM30a shRNA-transfected Jurkat cells. The cationic dye JC1 in functional, polarized mitochondria is aggregated and fluoresces red/ orange, whereas monomeric dye free in the cytoplasm fluoresces green. B, Flow cytometric analysis of JC-1 fluorescence in the stated concentration of Edelfosine. by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd.

calizing the native protein. We found a portion of the TMEM30a- port system affects both alkyl phospholipid uptake and their sen- GFP chimera was associated with the plasma membrane, and this sitivity to the drug (50, 51). location was the predominant one among cells with lower levels of As with the proapoptotic antitumor phospholipids, the proa- protein expression. We, and recently others (43), also found that in poptotic truncated phospholipids have an intracellular site of ac- cells expressing higher amounts of TMEM30a that a large portion tion. These products of free radical phospholipid oxidation are http://classic.jimmunol.org remained in the intracellular compartment in organelles identi- apoptotic components of oxidized low-density lipoproteins (32) ﬁed as endoplasmic reticulum, Golgi, and mitochondria. This in- that occur in the circulation of models of oxidative stress (52, 53), tracellular accumulation may have resulted from overexpression, after acute exposure to cigarette smoke (38), in developed ath- but the S. cerevisiae Lem3p-GFP ortholog is also associated both erosclerotic lesions (35), and in animals chronically ingesting with the plasma membrane and the endoplasmic reticulum (17), ethanol (36). Immunosuppressive oxidatively truncated phospho- where it participates in endocytic and exocytic processes, main- lipids are also formed by UVB irradiation of skin (54). The

Downloaded from tains amino phospholipid transmembrane asymmetry, and defines truncated Az-choline phospholipids (Az-PC) are derived from cell polarity (44–47). oxidative fragmentation of linoleoyl residues—the most common Edelfosine is a premier member of a small group of anticancer, polyunsaturated sn-2 fatty acyl residue—and are the most potent and anti-leishmenaisis, alkyl phospholipids that selectively reduce products that typify oxidatively truncated phospholipids. the viability of metabolically active, proliferating cells (1). Thus, One complication that obfuscates our understanding of the leukemic Jurkat cells, in contrast to resting PBLs, are sensitive to substrate selectivity of the lipid import systems is that the na- growth inhibition by alkyl choline phospholipids (9, 40). These ture of the fluorescent moiety affects phospholipid import (19). choline lipids have several targets, ranging from suppression of We found, however, that TMEM30a overexpression increased CTP:phosphocholine cytidylyltransfersase, and hence PC synthe- [3H]PAF uptake and that internalization of unlabeled Edelfosine sis (6), to modulation of several kinase signaling pathways (1) to or unlabeled Az-PC was reduced when TMEM30a was suppressed mitochondrial depolarization and apoptotic caspase activation (2). by shRNA. We can therefore be certain that transport substrate These targets are intracellular, and the sensitivity of various cells selection for these substrates was free from the interference of to Edelfosine is correlated with the extent to which they can ac- bulky fluorescent tags. cumulate the lipid (4, 10, 40, 42, 48, 49). Leishmania parasites Az-PC, or its alkyl homolog Az-LysoPAF, rapidly enters cells also express homologs of Lem3p, and manipulation of this im- and associates with respiring mitochondria (32). Thus, these lipid 3224 TMEM30a IN PHOSPHOLIPID IMPORT

oxidation products, even when exogenously presented, rapidly 3. Cuvillier, O., E. Mayhew, A. S. Janoff, and S. Spiegel. 1999. Liposomal ET-18- OCH(3) induces cytochrome c-mediated apoptosis independently of CD95 depolarize intracellular mitochondria in a way enhanced by Bid, (APO-1/Fas) signaling. Blood 94: 3583–3592. a proapoptotic member of the Bcl-2 family, and resisted by the 4. Mollinedo, F., J. L. Fernańdez-Luna, C. Gajate, B. Martıń-Martıń, A. Benito, R. Martıńez-Dalmau, and M. Modolell. 1997. Selective induction of apoptosis in antiapoptotic member Bcl-XL (31, 32). Evidence of rapid inter- cancer cells by the ether lipid ET-18-OCH3 (Edelfosine): molecular structure nalization of extracellular truncated phospholipids is confirmed requirements, cellular uptake, and protection by Bcl-2 and Bcl-XL. Cancer Res. by the protection against apoptosis afforded by overexpression of 57: 1320–1328. intracellular PAF acetylhydrolase (32), a phospholipase A that 5. Vrablic, A. S., C. D. Albright, C. N. Craciunescu, R. I. Salganik, and 2 S. H. Zeisel. 2001. Altered mitochondrial function and overgeneration of re- specifically hydrolyzes short chain phospholipids including PAF active oxygen species precede the induction of apoptosis by 1-O-octadecyl-2- and oxidatively truncated phospholipids (55, 56). Extracellular phos- methyl-rac-glycero-3-phosphocholine in p53-defective hepatocytes. FASEB J. 15: 1739–1744. pholipids therefore have ready access to intracellular organelles and 6. Boggs, K. P., C. O. Rock, and S. Jackowski. 1995. Lysophosphatidylcholine and receptors. 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine inhibit the CDP- Extension of this observation to the whole animal will have choline pathway of phosphatidylcholine synthesis at the CTP:phosphocholine cytidylyltransferase step. J. Biol. Chem. 270: 7757–7764. several consequences. First, one route to insensitivity to antitumor 7. Zaremberg, V., C. Gajate, L. M. Cacharro, F. Mollinedo, and C. R. 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by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. We conclude PCs and phosphatidylethanolamines with short 17. Kato, U., K. Emoto, C. Fredriksson, H. Nakamura, A. Ohta, T. Kobayashi, sn-2 residues (or fluorescently tagged phospholipids with increased K. Murakami-Murofushi, T. Kobayashi, and M. Umeda. 2002. A novel membrane protein, Ros3p, is required for phospholipid translocation across the plasma hydrophilicity) are readily accumulated by mammalian cells by membrane in Saccharomyces cerevisiae. J. Biol. Chem. 277: 37855–37862. a saturable, energy-dependent process that fully resembles that of 18. Riekhof, W. R., and D. R. Voelker. 2006. Uptake and utilization of lyso- S. cerevisiae. The protein TMEM30a is homologous, and indeed phosphatidylethanolamine by Saccharomyces cerevisiae. J. Biol. Chem. 281: 36588–36596. orthologous, to the lipid import system of this yeast, and prior work 19. Elvington, S. M., F. Bu, and J. W. Nichols. 2005. Fluorescent, acyl chain-labeled shows that mammalian TMEM30a interacts with the ATP8B1 phosphatidylcholine analogs reveal novel transport pathways across the plasma membrane of yeast. J. Biol. Chem. 280: 40957–40964. P-type ATPase (39) suggesting it functions as a heterodimeric com- 20. Katoh, Y., and M. Katoh. 2004. Identification and characterization of CDC50A, plex. Because uptake of choline—and not serine phospholipids— CDC50B and CDC50C genes in silico. Oncol. Rep. 12: 939–943. http://classic.jimmunol.org is enhanced by TMEM30a in the absence of ATP8B1 overexpres- 21. Braquet, P., L. Touqui, T. Y. Shen, and B. B. Vargaftig. 1987. Perspectives in platelet-activating factor research. Pharmacol. Rev. 39: 97–145. sion, TMEM30a may interact with members of the P4-ATPase 22. Ishii, S., T. Nagase, and T. Shimizu. 2002. Platelet-activating factor receptor. family. This import system is relevant because it provides access Prostaglandins Other Lipid Mediat. 68-69: 599–609. for extracellular lipids to alter intracellular lipid metabolism and 23. Prescott, S. M., G. A. Zimmerman, D. M. Stafforini, and T. M. McIntyre. 2000. Platelet-activating factor and related lipid mediators. Annu. Rev. Biochem. 69: for oxidatively truncated phospholipids to alter mitochondrial func- 419–445. tion and cell viability. 24. Ihida, K., D. Predescu, R. P. Czekay, and G. E. Palade. 1999. Platelet activating factor receptor (PAF-R) is found in a large endosomal compartment in human Downloaded from umbilical vein endothelial cells. J. Cell Sci. 112: 285–295. Acknowledgments 25. Zhu, T., F. Gobeil, A. Vazquez-Tello, M. Leduc, L. Rihakova, M. Bossolasco, We thank the Lerner Research Institute Imaging for support and providing G. Bkaily, K. Peri, D. R. Varma, R. Orvoine, and S. Chemtob. 2006. Intracrine DNA cores. We also thank Dr. G. K. Marathe for many helpful discussions. signaling through lipid mediators and their cognate nuclear G-protein–coupled receptors: a paradigm based on PGE2, PAF, and LPA1 receptors. Can. J. Physiol. Pharmacol. 84: 377–391. Disclosures 26. Bazan, N. G., S. P. Squinto, P. Braquet, T. Panetta, and V. L. Marcheselli. 1991. Platelet-activating factor and polyunsaturated fatty acids in cerebral ischemia or The authors have no financial conflicts of interest. convulsions: intracellular PAF-binding sites and activation of a fos/jun/AP-1 transcriptional signaling system. Lipids 26: 1236–1242. 27. Marrache, A. M., F. Gobeil, Jr., S. G. Bernier, J. Stankova, M. Rola- References Pleszczynski, S. Choufani, G. Bkaily, A. Bourdeau, M. G. Sirois, A. Vazquez- 1. van Blitterswijk, W. J., and M. Verheij. 2008. Anticancer alkylphospholipids: Tello, et al. 2002. Proinflammatory gene induction by platelet-activating factor mechanisms of action, cellular sensitivity and resistance, and clinical prospects. mediated via its cognate nuclear receptor. J. Immunol. 169: 6474–6481. Curr. Pharm. Des. 14: 2061–2074. 28. Melnikova, V. O., K. Balasubramanian, G. J. Villares, A. S. Dobroff, M. Zigler, 2. Gajate, C., A. M. Santos-Beneit, A. Macho, M. Lazaro, A. Hernandez-De Rojas, H. Wang, F. Petersson, J. E. Price, A. Schroit, V. G. Prieto, et al. 2009. Crosstalk M. Modolell, E. Munõz, and F. Mollinedo. 2000. Involvement of mitochondria between protease-activated receptor 1 and platelet-activating factor receptor and caspase-3 in ET-18-OCH(3)-induced apoptosis of human leukemic cells. Int. regulates melanoma cell adhesion molecule (MCAM/MUC18) expression and J. Cancer 86: 208–218. melanoma metastasis. J. Biol. Chem. 284: 28845–28855. The Journal of Immunology 3225

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