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Originally published In Press as doi:10.1074/jbc.M606100200 on November 2, 2006

J. Biol. Chem., Vol. 281, Issue 51, 39217-39224, December 22, 2006
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The Enzymatic Function of Tafazzin*

Yang Xu{ddagger}, Ashim Malhotra{ddagger}, Mindong Ren§, and Michael Schlame{ddagger}§1

From the Departments of {ddagger}Anesthesiology and §Cell Biology, New York University School of Medicine, New York, New York 10016

Received for publication, June 26, 2006 , and in revised form, October 24, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tafazzin is a putative enzyme that is involved in cardiolipin metabolism, it may carry mutations responsible for Barth syndrome. To identify the biochemical reaction catalyzed by tafazzin, we expressed the full-length isoform of Drosophila melanogaster tafazzin in a baculovirus-Sf9 insect cell system. Tafazzin expression induced a new enzymatic function in Sf9 cell mitochondria, namely 1-palmitoyl-2-[14C]linoleoyl-phosphatidylcholine:monolysocardiolipin linoleoyltransferase. We also found evidence for the reverse reaction, because tafazzin expression caused transfer of acyl groups from phospholipids to 1-[14C]palmitoyl-2-lyso-phosphatidylcholine. An affinity-purified tafazzin construct, tagged with the maltose-binding protein, catalyzed both forward and reverse transacylations between cardiolipin and phosphatidylcholine, but was unable to utilize CoA or acyl-CoA as substrates. Whereas tafazzin supported transacylations between various phospholipid-lysophospholipid pairs, it showed the highest rate for the phosphatidylcholine-cardiolipin transacylation. Transacylation activities were about 10-fold higher for linoleoyl groups than for oleoyl groups, and they were negligible for arachidonoyl groups. The data show that Drosophila tafazzin is a CoA-independent, acyl-specific phospholipid transacylase with substrate preference for cardiolipin and phosphatidylcholine.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations in the human tafazzin gene are the origin of Barth syndrome (1), a multisystemic disorder presenting with cardiomyopathy, skeletal myopathy, cyclic neutropenia, and growth retardation (2). However, little is known about the biological function of tafazzin. Tafazzin and its various homologs in the eukaryotic kingdom belong to a large superfamily of putative phospholipid acyltransferases (3). In human (4–7), yeast (8, 9), and fruit fly (10), deletion of tafazzin affects the deacylation-reacylation cycle that generates the mature fatty acid composition of cardiolipin (CL).2 This results in both a decrease of the CL concentration and a drastic change in its molecular composition (4–10). In addition, the concentration of monolysocardiolipin (MLCL) increases (9, 11), suggesting that tafazzin is involved in the reacylation step of the CL-MLCL remodeling cycle. The role of tafazzin in CL metabolism is consistent with its localization in mitochondria (12–14).

Although tafazzin has been implicated in CL remodeling, its actual catalytic function has not been established. Several members of the acyltransferase superfamily use phosphopantetheine-linked substrates, such as acyl-CoA or acyl-acyl carrier protein (3). On the other hand, yeast tafazzin has been shown to reacylate lysophosphatidylcholine (LPC) in the absence of acyl-CoA, albeit the acyl donor of this reaction could not be identified (13). Previously, we have shown that phospholipid transacylation is involved in CL remodeling in rat liver mitochondria, but the exact role of tafazzin in this pathway has remained obscure (15). The present study was undertaken to determine whether tafazzin is an acyltransferase, i.e. a catalytic activity that transfers acyl groups between substrates, and if so, to define its acyl donors and acyl acceptors.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Radiochemicals—1-Palmitoyl-2-[linoleoyl-1-14C]phosphatidylcholine (53 Ci/mol), 1-palmitoyl-2-[oleoyl-1-14C]phosphatidylcholine (56 Ci/mol), 1-stearoyl-2-[arachidonoyl-1-14C]phosphatidylcholine (56 Ci/mol), 1-palmitoyl-2-[linoleoyl-1-14C]phosphatidylethanolamine (53 Ci/mol), and 1-acyl-2-[arachidonoyl-1-14C]phosphatidylethanolamine (56 Ci/mol) were purchased from Amersham Biosciences. [1-14C]Linoleic acid (51 Ci/mol), 1-[palmitoyl-1-14C]-2-lyso-phosphatidylcholine (55 Ci/mol), and 1-[oleoyl-9,10-3H(N)]-2-lyso-phosphatidic acid (47 Ci/mmol) were purchased from PerkinElmer Life Sciences. Radioactivity was measured by liquid scintillation counting using Ecoscint from National Diagnostics.

Human Specimens—Cardiac tissue was excised from whole hearts after transplantation and stored at –80 °C. Lymphoblast cell lines were established by Epstein-Barr virus transformation of leukocytes isolated from whole blood using Ficoll-Hypaque gradients. The cell lines were cultured in RPMI 1640 medium in the presence of fetal bovine serum (10%), penicillin (50 international units/ml), and streptomycin (50 µg/ml) at 37 °C under 5% CO2 atmosphere. Patients with Barth syndrome had an established mutation in the tafazzin gene and presented with cardiomyopathy plus at least one non-cardiac symptom, such as neutropenia, growth retardation, or skeletal muscle weakness. Control patients had a normal tafazzin gene and presented either with idiopathic-dilated cardiomyopathy (heart tissue controls) or with unrelated neurological conditions (lymphoblast controls). Further details of the clinical protocols and approvals by institutional review boards, have been published (7).

Drosophila Strains—Tafazzin mutants of Drosophila melanogaster were generated by imprecise excision of the P element insert from fly stock y[1]; P{y[+mDint2] w[BR.E.BR] = SUPor-P}tafazzin[KG02529]/SM1; ry[506] (Bloomington Drosophila Stock Center) as described (10). Control flies were derived from the same strain by precise excision of the P element, which does not cause tafazzin deletion. A detailed characterization of the Drosophila model is given elsewhere (10). The gene encoding the mitochondrial acyl carrier protein was mutated in fly stock y[1] w[*]; P{w[+mC] = lacW}mtacp1[j4A6]/TM6B, Tb[+] because of insertion of a P element in the first exon. Because flies homozygous for this mutation were not viable after the larval stage, all analyses of CL compositions were done in larvae. Homozygous larvae were collected under a fluorescence microscope after the stock was rebalanced with TM6B, GFP balancer. All flies were maintained in 3-inch culture vials at 22 °C on a standard cornmeal-sucrose-yeast medium.

Baculovirus-Sf9 Cell Expression System—All reagents for baculovirus expression, including the Sf9 cell line and the Baculo-GoldTM TNM-FH insect medium, were purchased from BD Biosciences. Sf9 cells were cultured at 27 °C and passaged every 3 days. The cDNA encoding the full-length isoform of Drosophila tafazzin (dTAZ-RA) was inserted into the EcoRI site of the pAc-GHLT-A vector, designed to express an N-terminal GST fusion protein. Two million Sf9 cells were seeded per 60-mm cell culture dish and co-transfected with 5 µg of pAc-GHTL-A-dTAZ-RA DNA and 0.5 µg of linearized baculovirus DNA, using the BD BaculoGold transfection kit. Transfected cells were kept at 27 °C for 4 h, after which the transfection medium was removed and replaced by fresh TNM-FH medium. Transfected cells were cultured at 27 °C for several days and transfection efficiency of the supernatant was monitored. When sufficient transfection efficiency was reached, the supernatant was harvested and stored as GST-dTAZ-RA baculovirus stock solution at 4 °C until used in expression experiments. For tafazzin expression, Sf9 cells were infected with GST-dTAZ-RA baculovirus stock or with wild-type baculovirus stock as control. Cells were cultured at 27 °C for 3–4 days before isolation of subcellular fractions.

Isolation of Mitochondria and Microsomes—Yeast mitochondria (16) and lymphoblast mitochondria (17) were isolated as described. Mitochondria were isolated from adult D. melanogaster and from Sf9 cells by differential centrifugation at 4 °C. Whole animals or cells were homogenized with a tight-fitting Teflon glass homogenizer in isolation buffer containing 210 mM mannitol, 70 mM sucrose, 1 mM EGTA, and 5 mM Hepes (pH 7.2). Cell debris and nuclei were removed by centrifugation at 750 x g for 5 min. Mitochondria were collected from the supernatant by centrifugation at 17,000 x g for 20 min. Microsomes were collected from the post-mitochondrial supernatant by centrifugation at 100,000 x g for 60 min. Mitochondria and microsomes were washed, resuspended in a small volume of buffer, and stored at –80 °C. Protein concentrations of subcellular fractions were determined by the method of Lowry (18).

Western Blot Analysis—Proteins were denatured by boiling in 1x SDS loading buffer and then applied to 10% gels for SDS-PAGE. Protein bands were transferred onto a nitrocellulose membrane in a solution containing 20% methanol in Tris-glycine-SDS (Bio-Rad). After blocking, the blots were incubated overnight at 4 °C with 1 µg/ml of affinity-purified rabbit polyclonal antibodies raised against Drosophila tafazzin (10). This was followed by incubation with goat anti-rabbit antibody conjugated with horseradish peroxidase (1:3500; Bio-Rad) for 2 h at room temperature. SuperSignal (Pierce) was used as peroxidase substrate.

E. coli Expression and Purification of Tafazzin—The full-length cDNA of Drosophila tafazzin was inserted into the EcoRI-SaII site of the pMAL-c2 vector for expression of a protein construct consisting of tafazzin and the maltose-binding protein (MBP). BL21 E. coli cells were transformed with the vector and grown at 37 °C to an optical density of 0.6, after which expression of MBP-tafazzin was induced by addition of 1 mM isopropyl thiogalactoside. Incubation was continued for 2 h at 32 °C, and cells were harvested by centrifugation. Cell pellets were kept frozen until they were lysed by sonication in ice-cold buffer, containing 20 mM Tris (pH 7.4), 0.2 M NaCl, 1 mM EDTA, 10 mM beta-mercaptoethanol, 0.1% Triton X-100, and Roche protease inhibitor mixture. Membranes and cell debris were cleared from the lysate by centrifugation, and the supernatant was incubated with amylose resin for 1 h at 4°C to bind MBP-tafazzin. The resin was collected by centrifugation, washed, and then incubated in a medium containing 0.1 M Tris (pH 7.4), 0.01% Triton X-100, and protease inhibitor either in the absence or presence of 10 mM maltose to release MBP-tafazzin. The resin was removed by centrifugation, and the supernatant was stored at –80 °C for enzyme assays, SDS-PAGE with Coomassie Blue staining, and Western blots with the tafazzin antibody.

Lipid Analytical Methods—Lipids were extracted according to Bligh and Dyer (19). Phospholipids were separated by thinlayer chromatography on silica gel 60 plates and visualized by brief exposure to iodine vapor. Individual phospholipids were identified by comparison with commercial standard compounds. One-dimensional thin-layer chromatography was carried out with the solvent chloroform-methanol-water (65:25:4). Two-dimensional thin-layer chromatography was carried out with chloroform-methanol-20% ammonium hydroxide (65:30:5) in the first dimension and chloroform-acetone-methanol-acetic acid-water (50:20:10:10:5) in the second dimension. For fatty acid analysis, phospholipids were isolated by two-dimensional thin-layer chromatography. Silica gel spots were scraped off and treated overnight with 1 ml of 0.5 M HCl in methanol at 90 °C and subsequently neutralized with 1 ml of saturated sodium bicarbonate. Fatty acid methyl esters were extracted with hexane, treated with anhydrous sodium sulfate, dried, and reconstituted in a few microliters of hexane. The fatty acid composition was analyzed by gas chromatography at 185 °C using an SP 2330 capillary column (Supelco, Bellefonte, PA) and a flame ionization detector. For the analysis of molecular species, PC was isolated by two-dimensional thin-layer chromatography, extracted from silica into chloroform-methanol (2:3) and digested by 15 units of phospholipase C (Bacillus cereus) in a two-phase system consisting of diethyl etherethanol (95:5) and aqueous buffer (0.25 M Hepes, 0.03 M boric acid, pH 7.2). The produced diacylglycerol was derivatized with 3,5-dinitrobenzoylchloride and resolved by reversed-phase high-performance liquid chromatography on a Nucleosil C18 column (5-µm particles, 4.6 x 250-mm column dimension) developed with acetonitrile-2-propyl alcohol (4:1) at a flow rate of 1.0 ml/min (20). Fractions of 0.5 ml were collected, the solvent was evaporated, and radioactivity was measured by liquid scintillation counting. The composition of CL was analyzed by reversed-phase high-performance liquid chromatography with fluorescence detection. To this end, naphthyl-1-acetyl-CL dimethyl ester was produced and purified by solid-phase extraction as described before (21).

Transacylation Assays—The reaction mixture contained 0.1 µCi (2 nmol) of [14C]acyl-labeled phospholipid as acyl donor and 10 nmol of lysophospholipid as acyl acceptor in a total volume of 0.2 ml of buffer (10 mM 2-mercaptoethanol, 0.5 mM EDTA, 50 mM Tris, pH 7.3) equilibrated at 37 °C. Phospholipids were dried at the glass wall and re-dispersed in buffer using a sonicator bath. The reaction was started by addition of mitochondria (100 µg of protein) or MBP-tafazzin (3 µg) and terminated after 10 min by addition of 2 ml of methanol. For the measurement of endogenous transacylation activities in mitochondria from lymphoblasts, yeast, and fruit flies, the amount of mitochondrial protein was increased to 1.0 mg, 4 mM ADP was included in the reaction mixture, and the incubation time was extended to 30 min. Reactions were nearly linear under these conditions. After incubation, lipids were extracted, tracer phospholipids were added, and phospholipids were separated by two-dimensional thin-layer chromatography as described above. Phospholipids were visualized on silica with iodine vapor, and the silica spots were scraped off for the measurement of radioactivity. In some experiments, free fatty acids were recovered from the solvent front, and acyl-CoA was recovered from the aqueous phase after lipid extraction.

LPC Acyltransferase Assays—The reaction mixture contained 0.05 µCi (1 nmol) of 1-[14C]palmitoyl-2-lyso-PC in 0.2 ml of buffer (10 mM 2-mercaptoethanol, 0.5 mM EDTA, 50 mM Tris, pH 7.3) equilibrated at 37 °C. The reaction was started by addition of mitochondria (30 µg of protein) or MBP-tafazzin (3 µg), and it was stopped after 5 min by addition of 2 ml of methanol. In some experiments, acyl donors were added as indicated in the figure legends. When LPC:linoleoyl-CoA acyltransferase activity was measured in mitochondria, the reaction mixture also contained 50 µM linoleoyl-CoA, and the amount of protein was reduced to 5 µg. Reactions were nearly linear under these conditions. After incubation, lipids were extracted, PC was added as tracer, and phospholipids were separated by one-dimensional thin-layer chromatography as described above. PC spots were visualized on silica with iodine vapor and scraped off for the measurement of radioactivity.

Data Presentation—All measurements were performed in triplicate unless indicated otherwise. Data are expressed as means and S.E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Role of Phosphopantetheine-linked Acyl Groups—Tafazzin shares an acyltransferase motif with several enzymes that use phosphopantetheine-linked acyl groups as substrate, i.e. acyl-CoA and acyl-acyl carrier protein (3). We hypothesized that tafazzin catalyzes a similar reaction and expected to find a decrease in the incorporation of [14C]linoleoyl-CoA into phospholipids in patients with tafazzin deficiency. However, the incorporation of [14C]linoleoyl-CoA into PC, PE, and CL was severalfold increased in cardiac biopsies from patients with Barth syndrome (Table 1). Because this result did not confirm our hypothesis, we considered the possibility that tafazzin transfers acyl groups from or to the mitochondrial acyl carrier protein (mtACP). If this was the case, significant mutations of the mtACP should affect tafazzin function, including the remodeling of CL. However, we found that insertion of a P element into the first exon of the D. melanogaster mtACP gene, did not change the molecular composition of CL, even though the mutation was lethal at the larval stage. In contrast, mutation of the tafazzin gene resulted in alteration of the CL composition (Fig. 1). The data show that tafazzin but not mtACP is essential for CL remodeling in D. melanogaster. Overall, these results do not support the idea that phosphopantetheine-linked acyl groups are substrates of tafazzin.


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  		TABLE 1
Incorporation of [14C]linoleic acid into cardiac phospholipids

Homogenized human heart tissue (0.5 mg of protein) was incubated at 37 °C for 20 minutes in 0.2 ml of buffer, containing 50 mM Tris (pH 7.4), 0.5 mM EDTA, 10 mM 2-mercaptoethanol, 4 mM ATP, 4.5 mM MgCl2, 0.2 mM CoA, 0.1 µCi [14C]linoleic acid, and 30 nmol of lysophospholipid. Incorporation of radioactivity into the corresponding phospholipid was measured.

 


Figure 1
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  		FIGURE 1.
CL analysis in larvae of Drosophila mutants. Lipids were extracted from Drosophila larvae of a wild-type strain (WT), a tafazzin mutant ({Delta}TAZ), and a mtACP mutant ({Delta}mtACP). The naphthyl-1-acetyl derivative of CL dimethyl ester was formed and analyzed by high performance liquid chromatography with fluorescence detection (21). In each chromatogram, the fluorescence yield is plotted against the retention time from 15 to 35 min. Normal Drosophila CL has one major peak (marked by asterisk), in which molecular species contain palmitoleoyl and linoleoyl residues (22).

 
Phospholipid Transacylation in Isolated Mitochondria—Our previous study in rat liver mitochondria suggested involvement of phospholipid transacylation in cardiolipin remodeling (15). To investigate whether tafazzin is indeed a phospholipid transacylase, we measured acyl transfer from PC to MLCL in tafazzin-deficient mitochondria from human lymphoblasts, yeast, and fruit flies. Transacylation was reduced compared with the controls, but a significant residual activity remained (Fig. 2, upper panel). We also measured LPC acyltransferase activity in these mitochondria because Testet et al. (13) had shown that yeast tafazzin carries this function. The effect of tafazzin deficiency on LPC acyltransferase was similar to the effect on PC-CL transacylation (Fig. 2, lower panel).

The data shown in Fig. 2 are consistent with the idea that tafazzin can catalyze both LPC reacylation and PC-CL transacylation. However, clear-cut conclusions could not be drawn, because both activities remained detectable in tafazzin-deficient mitochondria. To boost the tafazzin activity relative to other acyltransferases, we utilized the baculovirus-Sf9 cell expression system. To this end, we transferred cDNA of the full-length Drosophila tafazzin (dTAZ-RA) into a baculovirus and expressed the recombinant baculovirus in Sf9 insect cells. This system produced GST-tagged tafazzin and a minor band with the size of untagged tafazzin, both of which were localized in mitochondria (Fig. 3A). Tafazzin expression altered the fatty acid composition of CL in Sf9 cells (Fig. 3B). At the same time, a novel transacylase activity was induced in Sf9 mitochondria, which transferred [14C]linoleoyl residues from PC to MLCL (Fig. 4A). Tafazzin expression also caused an increase of [14C]LPC acylation activity in the absence of acyl-CoA (Fig. 4B). In contrast, tafazzin expression did not change the activity of [14C]LPC:linoleoyl-CoA acyltransferase, which was 470 ± 78 pmol/min/mg protein in control Sf9 mitochondria and 478 ± 23 pmol/min/mg protein in tafazzin-expressing Sf9 mitochondria.


Figure 2
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  		FIGURE 2.
Endogenous mitochondrial acyl transfer activities in human lymphoblast, yeast, and fruit fly. Upper panel, formation of [14C]CL from 1-palmitoyl-2-[14C]acyl-PC and MLCL. The radiolabeled acyl group was [14C]oleoyl (for lymphoblasts and yeast) or [14C]linoleoyl (for fruit fly). Lower panel, formation of [14C]PC from 1-[14C]palmitoyl-2-lyso-PC. Closed bars represent control mitochondria and open bars represent tafazzin-deficient mitochondria.

 


Figure 3
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  		FIGURE 3.
Expression of Drosophila tafazzin in Sf9 cells. A, Western blot analysis. Subcellular fractions were isolated from Sf9 cells infected with wild-type baculovirus (BacV) or with recombinant baculovirus (TAZ-BacV). The recombinant virus encoded the full-length isoform of Drosophila tafazzin with GST tag, GST-dTAZ-RA. Western blot analysis was carried out with polyclonal antibodies raised against Drosophila tafazzin (10). The 80-kDa band corresponds to GST-tagged tafazzin; the 43-kDa band has the same size as untagged tafazzin. Numbers on the left represent molecular mass markers. B, fatty acid composition of phospholipids from Sf9 cells infected with wild-type baculovirus (BacV) or with recombinant baculovirus (TAZ-BacV).

 
The data shown in Fig. 4 suggest that Drosophila tafazzin is a phospholipid transacylase that uses PC as an acyl donor and MLCL as an acyl acceptor. The parallel expression of LPC acyltransferase activity gave the hint that this activity may be a reverse transacylation, with CL acting as acyl donor and LPC acting as acyl acceptor. To verify that CL can indeed donate acyl groups to [14C]LPC, we analyzed the effect of exogenous tetralinoleoyl-CL on the molecular composition of [14C]PC formed from [14C]LPC. When 1-[14C]palmitoyl-2-lyso-PC was incubated with tafazzin-expressing Sf9 mitochondria in the absence of exogenous acyl donors, radiolabeled PC was found in two peaks. Peak 1 contained 1-[14C]palmitoyl-2-palmitoleoyl-PC and peak 2 contained 1-[14C]palmitoyl-2-oleoyl-PC (Fig. 5A). This is consistent with the endogenous fatty acid composition of Sf9 cells, where palmitoleoyl and oleoyl make up 19 ± 2 and 52 ± 3% of all phospholipid-bound acyl groups, respectively. Upon addition of exogenous tetralinoleoyl-CL, peak 1, which also harbors 1-[14C]palmitoyl-2-linoleoyl-PC, increased at the expense of peak 2, suggesting that linoleoyl groups were transferred from CL to [14C]LPC (Fig. 5B).

Phospholipid Transacylation in Purified Tafazzin—To unequivocally identify substrates and products of the tafazzin reaction, we affinity-purified a tafazzin fusion protein. Because we encountered difficulties in the purification of GST-tagged tafazzin from Sf9 cells, we expressed an MBP-tagged tafazzin in E. coli and isolated it by affinity binding to amylose resin. This method yielded a nearly homogeneous preparation of the MBP-tafazzin fusion protein, which had high transacylase activity, measured either by acyl transfer from 1-palmitoyl-2-[14C]linoleoyl-PC to MLCL or by acyl transfer from CL to 1-[14C]palmitoyl-2-lyso-PC (Fig. 6A). The purified enzyme was able to convert [14C]LPC into [14C]PC only in the presence of CL but not in the presence of linoleoyl-CoA (Fig. 6B). Furthermore, addition of CoA did not stimulate the transfer of acyl groups from CL to [14C]LPC (Fig. 6B). These data demonstrate CoA-independent phospholipid transacylation by tafazzin. The purified enzyme transferred [14C]linoleoyl residues from [14C]PC to MLCL but not to CoA, forming [14C]CL but not [14C]linoleoyl-CoA (Fig. 6C). The formation of [14C]CL was strictly dependent on the presence of MLCL, and it was not affected by either CoA or ADP (Fig. 6C). Thus, the stimulating effect of ADP on CL remodeling, which we had observed in isolated mitochondria (15), was probably not mediated by tafazzin. Finally, purified tafazzin did not show phospholipase A2 activity, because it did not release [14C]linoleic acid from [14C]PC, even in the absence of an acyl acceptor (Fig. 6C).


Figure 4
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  		FIGURE 4.
Acyl transfer activities in Sf9 mitochondria expressing Drosophila tafazzin. Mitochondria were isolated from non-transfected Sf9 cells (NT), Sf9 cells infected with wild-type baculovirus (BacV), and Sf9 cells infected with recombinant baculovirus encoding Drosophila tafazzin (TAZ-BacV). A, formation of [14C]CL from 1-palmitoyl-2-[14C]linoleoyl-PC and MLCL. B, formation of [14C]PC from 1-[14C]palmitoyl-2-lyso-PC. C, formation of [3H]PA from 1-[3H]oleoyl-2-lyso-PA. Conditions were the same as for B, except that 1 µCi 1-[3H]oleoyl-2-lyso-PA was added instead of 1-[14C]palmitoyl-2-lyso-PC.

 


Figure 5
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  		FIGURE 5.
Molecular species of [14C]PC formed from 1-[14C]palmitoyl-2-lyso-PC. A, mitochondria (0.12 mg of protein) from Sf9 cells, expressing Drosophila tafazzin, were incubated with 0.2 µCi of 1-[14C]palmitoyl-2-lyso-PC in 0.8 ml of buffer containing 50 mM Tris (pH 7.4), 0.5 mM EDTA, and 10 mM 2-mercaptoethanol for 5 min at 37 °C. [14C]PC was isolated, derivatized, and separated by high performance liquid chromatography (20). Fractions of 0.5 ml were collected to measure radioactivity. Molecular species were identified by the retention time of standard compounds. Acyl groups are given as x:y, in which x specifies the number of carbon atoms and y the number of double bonds. Peak 1 contained both 16:0–16:1 and 16:0–18:2; the two species cannot be resolved by this technique (20). Peak 2 contained 16:0–18:1. B, mitochondria were incubated and processed as described in A. Bovine heart CL (18:2-CL, 50 µg) was added to the incubation mixture as exogenous acyl donor. C, peak 1/peak 2 radioactivity ratio in the presence of different exogenous acyl donors (50 µg each). 18:1-CL, tetraoleoyl-CL; 18:2-CL, bovine heart CL; 18:2-PC, 1-palmitoyl-2-linoleoyl-PC; 16:1-PC, dipalmitoleoyl-PC.

 


Figure 6
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  		FIGURE 6.
Transacylation by purified MBP-tafazzin. A, MBP-tafazzin (85 kDa) was expressed in E. coli, solubilized, and loaded on amylose resin. The resin was washed, and proteins were released either in the absence (–) or in the presence (+) of 10 mM maltose. Released proteins were analyzed by SDS-PAGE followed by Coomassie Blue staining (CB) or by Western blot analysis with the tafazzin antibody (WB). Molecular mass markers are shown on the left. Transacylation was measured as acyl transfer from bovine heart CL to 1-[14C]palmitoyl-2-lyso-PC (open columns) and as acyl transfer from 1-palmitoyl-2-[14C]linoleoyl-PC to MLCL (closed columns). B, conversion of 1-[14C]palmitoyl-2-lyso-PC to [14C]PC by affinity-purified MBP-tafazzin in the presence of different co-substrates, including 50 µM linoleoyl-CoA (18:2-CoA), 50 µM bovine heart CL, and 200 µM CoA. C, transfer of [14C]linoleoyl groups from 1-palmitoyl-2-[14C]linoleoyl-PC to CL, free fatty acids (FA), and acyl-CoA by affinity purified MBP-tafazzin in the presence of various co-substrates, including 200 µM CoA, 50 µM MLCL, and 4 mM ADP.

 
Substrate Specificity of Drosophila Tafazzin—To study the substrate specificity of Drosophila tafazzin, we incubated Sf9 mitochondria from tafazzin-expressing cells with various pairs of acyl donors and acyl acceptors. The transfer rate of linoleoyl groups from PC to MLCL was about 10-fold higher than the transfer rate of oleoyl groups, which in turn was about twice as high as the transfer rate of arachidonoyl groups (Fig. 7). The low activity of oleoyl substrates compared with linoleoyl substrates was also seen in the reverse reaction, because tetraoleoyl-CL did not donate acyl groups to [14C]LPC as effectively as tetralinoleoyl-CL (Fig. 5C). Drosophila tafazzin also catalyzed transacylations between PE and CL, between PC and PE, and between PC and PA, although these activities were much lower than the PC-CL transacylation (Fig. 7). As expected, these transacylations were reversible. For instance, we found similar acyl transfer rates with the substrate pairs [14C]PC/LPE and LPC/[14C]PE (Fig. 7). Palmitoleoyl groups were also transferred from exogenous PC to [14C]LPC (Fig. 5C), suggesting that tafazzin can reshuffle molecular species within a single phospholipid class. Transfer of palmitoleoyl groups by Drosophila tafazzin is consistent with the high abundance of palmitoleoyl in Drosophila CL (22). To corroborate the fact that LPA is a substrate of Drosophila tafazzin, we incubated 1-[3H]oleoyl-2-lyso-PA with Sf9 mitochondria. The effect of tafazzin expression on [3H]LPA acylation was similar to the effect on [14C]LPC acylation, suggesting that tafazzin can transfer acyl groups from endogenous mitochondrial phospholipids to [3H]LPA (Fig. 4C).


Figure 7
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  		FIGURE 7.
Substrate specificity of Drosophila tafazzin. Mitochondria from tafazzin-expressing Sf9 cells were incubated with various substrate pairs, each consisting of a 2-[14C]acyl-phospholipid and an unlabeled lysophospholipid. Transfer of the [14C]acyl group to the lysophospholipid was measured. Transacylation rates were corrected for the background activity measured in Sf9 mitochondria that do not express Drosophila tafazzin. Rates are presented as percent of the rate measured with the substrate pair [14C]18:2-PC and MLCL. 18:1, oleoyl group; 18:2, linoleoyl group; 20:4, arachidonoyl group.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this article, we demonstrate that full-length tafazzin from D. melanogaster is a phospholipid transacylase. When expressed in Sf9 cells, the enzyme gives raise to a new catalytic activity, which is the transfer of linoleoyl residues from PC to MLCL. This activity is not present in control Sf9 mitochondria (non-transfected control and wild-type-baculovirus control), either because the endogenous tafazzin activity is too low, or because Sf9 tafazzin has an acyl specificity that does not allow the transfer of linoleoyl groups (linoleoyl groups are absent from Sf9 phospholipids). The transacylation is reversible, converting LPC to PC with acyl groups derived from CL, PE, or PC. In addition, tafazzin catalyzes acyl transfer from PE to MLCL and from PC to LPA, suggesting that CL, PC, PE, PA, and perhaps other phospholipids are substrates of this enzyme. However, the activity is highest with the PC/MLCL substrate pair. Transacylase activity was confirmed in affinity-purified MBP-tafazzin. In the purified enzyme, we could show directly that acyl-CoA is not an acyl donor, and CoA is not an acyl acceptor of tafazzin.

It was surprising to find transacylase activity in tafazzin, because tafazzin shares conserved motifs with several enzymes that operate with phosphopantetheine-linked substrates (3). However, the present data do not support any involvement of phosphopantetheine-linked acyl groups in CL remodeling, and they clearly show that Drosophila tafazzin does not possess LPC:linoleoyl-CoA acyltransferase activity. Phospholipid transacylations have been implicated in a number of cellular functions, although in most instances, the responsible enzymes have not been characterized (see Ref. 23 for a review). For example, phospholipid transacylation is involved in the formation of N-acyl-PE (24), bis-(monoacylglycero)-phosphate (25), and plasmenylethanolamine (26). Transacylases are generally thought to be phospholipases that allow acyl transfer from an active enzyme-acyl intermediate back to a lysophospholipid, as an alternative pathway to hydrolysis (23). This type of transacylation is a ping-pong reaction, in which the donor phospholipid and the acceptor lysophospholipid alternate at the active site. However, tafazzin does not follow this mechanism, because it does not possess any phospholipase activity, even in the absence of acyl acceptors, suggesting that the reaction does not proceed via a free enzyme-acyl intermediate. Furthermore, tafazzin is not homologous to phospholipases, but rather contains the HX4D motif that is highly conserved throughout the acyltransferase superfamily (27). The invariant histidine in this motif is thought to deprotonate the sn-2 hydroxyl group of lysophospholipids, thereby facilitating a nucleophilic attack on the ester bond of the acyl donor (28). If this model does indeed apply to the tafazzincatalyzed transacylation, the active intermediate must be a lysophospholipid-phospholipid complex attached to the active site of the enzyme.


Figure 8
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  		FIGURE 8.
Postulated CL remodeling pathway. The enzymes of this pathway are phospholipase A, tafazzin (E), and LPC:acyl-CoA acyltransferase. Although the tafazzin reaction is reversible, the mass effect of the two other reactions drives it toward the left side of the equation (formation of CL and LPC). The net reaction of the pathway is the hydrolysis of acyl-CoA, which has a negative change in free energy, providing the driving force. Acyl specificity is conferred by tafazzin.

 
The phospholipid specificity of Drosophila tafazzin is consistent with its role in CL remodeling, and the acyl specificity is consistent with the molecular composition of Drosophila CL, where linoleoyl and palmitoleoyl groups dominate (22). However, transacylations are near-equilibrium reactions that, for thermodynamic reasons, cannot facilitate unidirectional transfer of specific acyl residues from one phospholipid to another. To provide net transfer of fatty acids, transacylations have to be coupled to irreversible chemical reactions. Such irreversible reactions may hypothetically include the deacylation of CL and the reacylation of LPC by acyl-CoA, because these reactions convert the products of tafazzin back to its substrates (Fig. 8). According to this postulate, the net reaction of CL remodeling is the hydrolysis of acyl-CoA, which has a negative change in free energy and is thus capable to drive the remodeling process (Fig. 8). The scheme in Fig. 8 also makes reference to a number of previous observations. First, the MLCL concentration was shown to increase in tafazzin-deficient cells (9, 11), suggesting that the deacylation of CL is independent of tafazzin. Second, tafazzin deficiency affected the molecular composition of PC (7, 29), suggesting that tafazzin plays an active role in PC remodeling. Third, CL remodeling was shown to involve acyl-selective inflow of fatty acids and non-selective outflow of fatty acids (15). This is consistent with our model in which only tafazzin has strong acyl specificity (Fig. 8).

In conclusion, tafazzin is an acyl-specific transacylase that is crucial for CL remodeling. It may catalyze both the acylation of LPC with CL-derived acyl groups and the acylation of MLCL with PC-derived acyl groups. To our knowledge, tafazzin is the first CoA-independent phospholipid transacylase that has been identified. Tafazzin may also reshuffle the molecular composition of other phospholipids, because it is able to shuttle specific acyl groups between various phospholipid classes and even between different species of a single phospholipid class. This raises the question of whether multiple transcripts of the tafazzin gene, which have been identified in human (30, 31), mouse (31), and fruit fly (10), have in fact specific functions in different phospholipid pathways. If tafazzin does indeed have multiple functions, they may affect more than one intracellular site, because the enzyme was recently found in both the inner and outer mitochondrial membrane (32).


   FOOTNOTES
 
* This work was supported in part by grants from the National Institutes of Health (1 R01 HL078788-01 A1, to M. S.), the American Heart Association (0350126N, to M. S.), and the Barth Syndrome Foundation (to Y. X. and M. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Anesthesiology, NYU School of Medicine, 550 First Ave., NY, NY 10016. Tel.: 212-2630648; Fax: 212-2636139; E-mail: michael.schlame{at}med.nyu.edu.

2 The abbreviations used are: CL, cardiolipin; GST, glutathione S-transferase; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; MBP, maltose-binding protein; MLCL, monolysocardiolipin; mtACP, mitochondrial acyl carrier protein; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Jeffrey A. Towbin for supplying human heart samples, Dr. Richard I. Kelley for supplying human lymphoblast cell lines, and Dr. Miriam L. Greenberg for providing yeast mitochondria from wild-type and tafazzin-deficient strains. We also acknowledge advice and technical support from Dr. Jin Zhang, Dr. Jie Li, and Dr. Pavithra Ranganathan.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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