The Bifunctional Protein TtFARAT from Tetrahymena thermophila Catalyzes the Formation of both Precursors Required to Initiate Ether Lipid Biosynthesis*

Background: Fatty acyl reductases (FARs) are monofunctional proteins of similar size and domain structure. Results: The only FAR present in Tetrahymena thermophila is fused with a dihydroxyacetone phosphate acyltransferase and localized in the peroxisomes. Conclusion: T. thermophila FAR-like protein is a bifunctional protein resulting from a gene fusion event. Significance: T. thermophila FAR-like protein provides both substrates required to initiate ether lipid biosynthesis. The biosynthesis of ether lipids and wax esters requires as precursors fatty alcohols, which are synthesized by fatty acyl reductases (FARs). The presence of ether glycerolipids as well as branched wax esters has been reported in several free-living ciliate protozoa. In the genome of Tetrahymena thermophila, the only ORF sharing similarities with FARs is fused to an acyltransferase-like domain, whereas, in most other organisms, FARs are monofunctional proteins of similar size and domain structure. Here, we used heterologous expression in plant and yeast to functionally characterize the activities catalyzed by this protozoan protein. Transient expression in tobacco epidermis of a truncated form fused to the green fluorescence protein followed by confocal microscopy analysis suggested peroxisomal localization. In vivo approaches conducted in yeast indicated that the N-terminal FAR-like domain produced both 16:0 and 18:0 fatty alcohols, whereas the C-terminal acyltransferase-like domain was able to rescue the lethal phenotype of the yeast double mutant gat1Δ gat2Δ. Using in vitro approaches, we further demonstrated that this domain is a dihydroxyacetone phosphate acyltransferase that uses preferentially 16:0-coenzyme A as an acyl donor. Finally, coexpression in yeast with the alkyl-dihydroxyacetone phosphate synthase from T. thermophila resulted the detection of various glycerolipids with an ether bond, indicating reconstitution of the ether lipid biosynthetic pathway. Together, these results demonstrate that this FAR-like protein is peroxisomal and bifunctional, providing both substrates required by alkyl-dihydroxyacetone phosphate synthase to initiate ether lipid biosynthesis.

The biosynthesis of ether lipids and wax esters requires as precursors fatty alcohols, which are synthesized by fatty acyl reductases (FARs). The presence of ether glycerolipids as well as branched wax esters has been reported in several free-living ciliate protozoa. In the genome of Tetrahymena thermophila, the only ORF sharing similarities with FARs is fused to an acyltransferase-like domain, whereas, in most other organisms, FARs are monofunctional proteins of similar size and domain structure. Here, we used heterologous expression in plant and yeast to functionally characterize the activities catalyzed by this protozoan protein. Transient expression in tobacco epidermis of a truncated form fused to the green fluorescence protein followed by confocal microscopy analysis suggested peroxisomal localization. In vivo approaches conducted in yeast indicated that the N-terminal FAR-like domain produced both 16:0 and 18:0 fatty alcohols, whereas the C-terminal acyltransferase-like domain was able to rescue the lethal phenotype of the yeast double mutant gat1⌬ gat2⌬. Using in vitro approaches, we further demonstrated that this domain is a dihydroxyacetone phosphate acyltransferase that uses preferentially 16:0-coenzyme A as an acyl donor. Finally, coexpression in yeast with the alkyl-dihydroxyacetone phosphate synthase from T. thermophila resulted the detection of various glycerolipids with an ether bond, indicating reconstitution of the ether lipid biosynthetic pathway. Together, these results demonstrate that this FAR-like protein is peroxisomal and bifunctional, providing both substrates required by alkyl-dihydroxyacetone phosphate synthase to initiate ether lipid biosynthesis.
Primary fatty alcohols are found throughout the biological world, present either as free fatty alcohols or, most commonly, as component of complex molecules such as ether lipids or wax esters. Wax esters are present in all life kingdoms, serving as energy storage in some bacteria and protists, as pheromone signals in insects, as factors controlling buoyancy in deep-diving marine species, as lipid constituents of the waterproofing barriers in plants (cuticle) and animals (sebum and meibum), or as cleansing and lubricating agents of feathers in birds (1)(2)(3)(4)(5)(6)(7). Ether glycerolipids are widely spread among marine and land animals but almost absent in plant cells (8). They are ubiquitous constituents of animal cell membranes, and derivatives such as plasmalogens and platelet-activating factor have critical functions in human health (8). Rhyzomelic chondrodysplasia punctata, a pathology resulting from deficiencies in ether lipid biosynthesis, leads to death in infancy, underlining the important role of ether lipids in normal development and function of specialized cells, for example for the myelinization process of nervous cells in the brain (9). Fatty alcohols are synthesized by fatty acyl reductases (FARs) 2 that catalyze a two-step, four-electron reduction via an unreleased aldehyde intermediate and using NAD(P)H as a reducing equivalent (10). Microsomal and plastidial FARs, using as substrates acyl-CoAs or acyl-acyl carrier proteins, respectively, have been characterized (11). Genes coding for FARs have been isolated from bacteria, plants, insects, and animals, and all encoded proteins are of similar size (50 -60 kDa) and structural domain organization. FAR proteins have a Rossmann fold domain at the N terminus for NAD(P)H binding and a fatty acyl-CoA reductase (FAR_C) domain at the C terminus (11). In mammals, two FARs highly expressed in tissues known to accumulate significant amounts of wax esters, like the skin and the eyelid, or ether lipids, like the brain, have been identified (12). They both possess a C-terminal extension of 66 amino acids potentially representing a transmembrane domain that is responsible for their targeting to peroxisomes (13). Plant and insect FARs are devoid of such a hydrophobic domain and may be soluble or associated with the endoplasmic reticulum (3,11).
Following their synthesis, primary fatty alcohols are used as acyl acceptors by wax synthases to produce wax esters in the endoplasmic reticulum or to replace the acyl-chain of sn-1acyl-dihydroxyacetone phosphate by alkyl-dihydroxyacetone phosphate synthase (ADPS) to generate an ether bond linkage in the peroxisomes (5,8). In the latter case, acylation of DHAP with an acyl-CoA by the peroxisomal DHAP acyltransferase (DHAPAT) is considered the initial step of ether lipid biosynthesis (8). Because wax synthase enzymes also belong to the acyltransferase (AT) superfamily, both wax ester and ether lipid biosynthesis rely on FAR and AT activities.
Tetrahymena species are unicellular ciliate protozoa that have been used as models for molecular and cellular biology for decades, especially for studying lipid composition changes in response to modification of the environmental temperature (14). Tetrahymena pyriformis is characterized by the presence of high amounts of ether glycerolipids that are especially enriched in phosphatidylcholine and 2-aminoethyl-phosphonolipid (15). The presence of smaller amounts of wax esters in T. pyriformis enriched in branched fatty acids and alcohols has also been reported (16).
Using the genomic resource generated from the sequencing of the macronucleus from Tetrahymena thermophila (17), we report here the functional characterization of the unique ORF coding for a FAR present in the genome of this organism. The corresponding protein contains apparently not only a putative FAR domain but also an acyltransferase-like domain at its C terminus, whereas, in most organisms, FARs are monofunctional proteins. Using heterologous expression in plant and yeast together with in vitro assays, we show that this protein localizes in the peroxisomes and is bifunctional with its N-terminal end carrying FAR activity, whereas its C-terminal end displays DHAPAT activity. Its coexpression with T. thermophila ADPS resulted in implementing ether lipid biosynthesis in yeast, suggesting that this FAR-like protein provides both substrates required by ADPS to initiate ether lipid biosynthesis in the peroxisomes. Construction of Yeast Expression Vectors-Because of the unusual genetic code of Tetrahymena, the TtFARAT and TtAGPS genes were synthesized by Genscript with codon optimization for yeast expression and appropriate flanking sequences and restriction sites. TtFARAT was transferred from Genscript pUC57-TtFARAT into the yeast expression vector pVT-LEU by ligation using BamHI and XhoI restriction sites, yielding pVTLEU-TtFARAT. TtFAR and TtAT were amplified by PCR using pUC57-TtFARAT as a template and pairs of primers containing the attB1 and attB2 flanking sequences for Gateway recombinational cloning technology (18) as follows: far-f (GGGGACAAGTTTGTACAAAAAAGCA GGCTGGAT-CCACATAATGGGAAAGGTTTTCCAATTCTACGAAGG-AAAGACTGTTTTGTTGACTGG) with far-r (GGGACCAC-TTTGTACAAG AAAGCTGGGTACTCGAGTTACTTGAA-TGGCTTTCCAGAAGACAAAGCCCAGTTGATATCAGA-GAAGTATGGG) and at-f (GGGGACAAGTTTGTACAAAA-AAGCAGGCTGGATCCACATAATGCCAAGATCTTACG-AAGAATACAAATCTTTGTTGTTC) with at-r (GGGACCA-CTTTGTACAAG AAAGCTGGGTACTCGAGCTACAATC-TAGCCAAAATTGGATATTCAGAC) for amplifying TtFAR and TtAT, respectively. PCR products were first cloned into the pDONR221 ENTRY vector by BP cloning, generating pDONR221-TtFAR and pDONR221-TtAT. Open reading frames were subsequently transferred into the yeast expression vector pVT-LEU-GW by LR cloning, yielding pVTLEU-TtFAR and pVTLEU-TtAT. The pVT-LEU-GW vector was generated from pVT102-U-GW (19) by replacing its uracil selection cassette with the leucine selection cassette from pESC-LEU (Invitrogen) using PCR amplification and BglII restriction sites. TtADPS was subcloned directly into the yeast expression vector pVT102-U-GW by LR cloning using Genscript pUC57-TtADPS as the ENTRY clone.

Materials-All
Tobacco Transient Expression and Confocal Microscopy-The plant binary vector expressing the GFP-TtAT fusion protein was generated by LR cloning using pDONR221-TtAT and the pK7WGF2 destination vector (18). Constructs were transferred into the Agrobacterium tumefaciens GV3101 strain and used for transient expression in tobacco leaves, as described previously (20). Four-week-old tobacco (Nicotiana tabacum cv. Petit Havana) greenhouse plants grown at 22-24°C were used for transient expression. Transformed leaves were analyzed 48 h after infection of the lower epidermis. Confocal imaging was performed using a Leica TCS SP2 confocal laser-scanning microscope with a ϫ63 oil immersion objective. For imaging GFP and red fluorescent protein (RFP) constructs, excitation lines of an argon ion laser of 488 or 543 nm were used alternately with line switching using the multitrack facilities of the microscope.
Yeasts Expression and Complementation-The Saccharomyces cerevisiae wild-type INVSc1 strain (MATa his3⌬1 leu2 trp1-289 ura3-52) was used for the functional characterization of TtFARAT, TtFAR, and TtAT. The cmy228 strain (gat1⌬ gat2⌬ ϩ (pGAL1::GAT1(URA3))) (21) was used for complementation studies, yielding the cmyFARAT strain (gat1⌬ gat2⌬ ϩ (pADH::FARAT(LEU2))). The various yeast strains were transformed by a polyethylene glycol/lithium acetate protocol and selected on minimal medium agar plates lacking the corresponding amino acids. The INVSc1 strain was transformed with different pVT-LEU constructs, and transformants were selected on minimal medium agar plates lacking leucine. The cmy228 strain was transformed with the same pVT-LEU constructs, but transformants were selected on minimal medium agar plates lacking uracil and leucine and containing galactose as a unique sugar source. Complementation was tested on medium lacking uracil and leucine but containing glucose as a sugar source, and contraselection to discard the pGAL1::GAT1(URA3) episome was achieved in the presence of 1 mg/ml 5-fluoroorotate. The elimination of ScGAT1 and the presence of TtFARAT or TtAT were verified by PCR using the following primers: ATACGAAGGGCTGTGTAG and TCAA-CACCGATTTCACCG for ScGAT1, CGTTAACTCTGA-TAAGAGAGGTTGG and CGTATTCGTAGAAGATAGCC for TtFARAT, and CCCAGATGCTAAGATCGTTCC and CAGCACCAGACTTATCAGC for TtAT. All transgenic yeast expressions were carried for 2 days in 5-25 ml liquid minimal medium lacking leucine and/or uracil.
Total Fatty Acyl Chain Analyses-Usually, 5 ml of yeast cell cultures were harvested by centrifugation, and the supernatant was poured into a separate tube. When analyzed, the medium was extracted twice with 2 ml of 2:1 chloroform:methanol and once with 2 ml of chloroform. The organic phases were combined and washed with 2.5 ml of 0.9% NaCl (w/v) before being evaporated under a gentle stream of nitrogen. Fatty acid methyl esters were obtained by transmethylation at 85°C for 1 h with 0.5 M sulfuric acid in methanol containing 2% (v/v) dimethoxypropane and 50 mg of heptadecanoic acid (C17:0) as well as 20 mg of pentadecanol (C15:0-OH) as internal standards. After cooling, 1 ml of NaCl (2.5%, w/v) was added, and fatty acyl chains were extracted twice with 2 ml dichloromethane. Extracts were dried under a gentle stream of nitrogen and dissolved into 150 l of N,O-bis(trimethylsilyl) trifluoroacetamide:trimethylchlorosilane (99:1), and free hydroxyl groups were derivatized at 110°C for 15 min. Surplus N,O-bis(trimethylsilyl) trifluoroacetamide:trimethylchlorosilane was evaporated under nitrogen, and samples were dissolved in a 1:1 (v/v) mixture of hexane:toluene. Samples were subsequently analyzed by GC-MS as described previously by Domergue et al. (19).
Extraction of Lipids and Separation-Lipid analysis of transgenic yeast cells were made from 25-ml cultures grown for 48 h at 30°C. Cells were harvested by centrifugation, washed with 10 ml of NaCl 2.5% (w/v), and then the lipids were extracted successively with 2 ml of chloroform/methanol (1:1), 2 ml of chloroform/methanol (2:1), and 2 ml of chloroform. The resulting organic phase was extracted with 2 ml of 2.5% NaCl (w/v), dried on hydrophilic cotton, and evaporated under a gentle stream of nitrogen. The residue was dissolved in 1 ml of chloroform, and polar and neutral lipids were separated using solid phase extraction on an Upti-Clean SI-S-100/1 column (Interchim). Neutral lipids were eluted with chloroform, whereas methanol was used for the elution of polar lipids. The polar lipid fractions were further analyzed by thin-layer chromatography using high-performance thin layer chromatography (HPTLC) Silica Gel 60 plates (Merck) and methyl acetate/propanol/chloroform/methanol/KCl 0.25% (25:25:25:10:9, v/v/v/v/v) as solvent, allowing the isolation of the major phospholipids phosphatidylcholine, phosphatidylinositol and phosphatidylserine, and phosphatidylethanolamine. The acyl chain composition of these differ- and heptadecanoic acid (C17:0) were used as internal standards (IS). Peaks indicated by an asterisk correspond to indole derivatives. B, total fatty acyl chain quantification. Yeast cultures were grown for 48 h at 30°C. Lipids were transmethylated, and free hydroxyl groups were derivatized to trimethylsilyl ethers before separation by gas chromatography and detection by mass spectrometry. The amounts of total fatty acyl chains (intracellular and secreted) produced are expressed in micrograms/A 600 unit with standard deviation (n ϭ 4). ent lipid classes was then analyzed by GC-MS using the same protocol as for the total fatty acyl chain analysis.

RESULTS
The Tetrahymena FAR Candidate Contains Two Domains and Is Likely Localized in Peroxisomes-Using the Arabidopsis FAR3/CER4 sequence (26) as a query, a search of the T. thermophila genome database revealed a unique ORF of 3423 bp, TTHERM_00221020, coding for a protein of 1140 amino acids (GenBank TM accession number XP_001020674.1). The size of the encoded protein clearly differed from all FARs isolated so far in various kingdoms, which were systematically of about 450 -550 amino acids. The first 480 amino acids, which contained both the NAD_binding_4 (PF07993) and male sterility (PF3015) domains, led to its original annotation as male sterility protein. Nevertheless, if this N-terminal extremity is most similar to the human fatty acyl reductase 1 (HsFAR1) protein, its C-terminal 660 amino acids include the acyltransferase motif PF01553, and it is most related to the human glyceronephosphate-O-acyltransferase (Fig. 1A, HsGNPAT). Because male sterility domains are also annotated in databases as FAR_C, a signature for FAR (11) and because glyceronephosphate-Oacyltransferases belong to the AT superfamily, we decided to rename this protein TtFARAT and to undertake its functional characterization.
Sequence analysis programs on the basis of hydropathy plots gave conflicting results regarding the presence or absence of classical transmembrane domains within TtFARAT. In contrast, the presence of a type 1 peroxisomal targeting signal (ARL) at the C-terminal end of the TtFARAT protein was recognized systematically. To establish the subcellular localization of TtFARAT, we generated the fluorescent fusion protein GFP-TtAT, where the N-terminal FAR domain was replaced by green fluorescent protein. The corresponding binary vector was transiently expressed in tobacco leaf epidermal cells together with the peroxisomal marker px-RFP (27). Confocal microscopy analysis showed that the GFP-TtAT fusion protein colocalized with the peroxisomal marker (Fig. 1B), indicating that TtFARAT is most probably localized in the peroxisomes.
TtFARAT Produces Fatty Alcohols and Complements a Yeast Acyltransferase Mutant-The functional characterization of TtFARAT as well as of each of its domains on its own (i.e. TtFAR and TtAT) was achieved by heterologous expression in yeast. GC-MS analyses indicated that TtFARAT expression resulted in the production of high levels of hexadecanol (16:0-OH) and octadecanol (18:0-OH) in the cells as well as in the medium ( Fig. 2A). In total, about 2 g of fatty alcohols per unit A were produced by TtFARAT after 48 h of expression (Fig. 2B). A similar analysis showed that expression of the TtAT domain did not led to any difference in total fatty acyl profile when compared with a control (Fig. 2B). In contrast, the expression of the TtFAR domain alone was sufficient to produce the same fatty alcohols with a 66% higher yield (Fig. 2B), indicating that TtFAR is, per se, a fatty acyl reductase.
To genetically assess the acyltransferase activity of TtFARAT, its ability to rescue the lethal phenotype of the yeast double mutant gat1⌬ gat2⌬ was assessed using plasmid shuffle complementation studies. In yeast, GAT1 and GAT2 are the only two acyltrans-ferases acylating the sn-1 position of G 3 P and DHAP, thus being essential to initiate glycerolipid biosynthesis. The cmy228 mutant (gat1⌬ gat2⌬ ϩ (pGAL1::GAT1(URA3))) bears chromosomal deletions of both GAT1 and GAT2 but contains an episomal GAT1 gene expressed under the control of the inducible GAL1 promoter. Therefore, it remains viable in the presence of galactose but does not grow on glucose-containing medium. The cmy228 strain was transformed with the empty vector (pVTLEU) as well as with the same vector containing TtFARAT, TtFAR, or TtAT under the constitutive promoter pADH (pVTLEU-TtFARAT, pVTLEU-TtFAR, or pVTLEU-TtAT, respectively). Several transformants were selected on inducible medium (ura-/leu-/GAL) and thereafter transferred to glucose-containing medium (ura-/leu-/GLU) to repress GAT1 expression (Fig. 3A). Although transformants carrying the empty vector control or pVTLEU-TtFAR failed to give colonies on this selective medium, those expressing TtFARAT or TtAT grew. After several rounds of counterselection on medium containing 5Ј-fluoroorotic acid to discard the pGAL1::GAT1(URA3) episome, these transformants remained viable on medium lacking leucine (leu-/ FIGURE 3. TtAT rescued the lethal phenotype of the cmy228 mutant strain. A, the cmy228 mutant (gat1⌬ gat2⌬ ϩ (pGAL1::GAT1(URA3))) was transformed with pVTLEU-TtFARAT, pVTLEU-TtFAR, or pVTLEU-TtAT or with the corresponding empty vector (pVTLEU) as control, and four transformants were selected on inducible medium (ura-/leu-/GAL). Complementation was assayed on glucose-containing medium (ura-/leu-/GLU). B, after several rounds of counter-selection on medium containing 5Ј-fluoroorotic acid, lost of the pGAL1::GAT1(URA3) episome was assayed on medium lacking leucine (leu-/GLU) and leucine and uracil (ura-/leu-/GLU). C, the absence of GAT1 and presence of TtFARAT or TtAT were confirmed by PCR analysis.
GLU) but were no longer able to grow on medium lacking leucine and uracil (Fig. 3B, ura-/leu-/GLU), indicating loss of the GAT1 episome. The absence of GAT1 and the presence of TtFARAT or TtAT were then confirmed by PCR analysis using primers specific to GAT1, TtFARAT, or TtAT (Fig. 3C). Altogether, these results indicate that the TtFARAT protein is bifunctional, its TtFAR domain being a fatty acyl reductase capable of reducing both palmitic and stearic acyl chains to the corresponding fatty alcohols, whereas its TtAT domain has GPAT and/or DHAPAT activity.
TtFARAT Preferentially Displays DHAP Acyltransferase Activity-To complete the functional characterization of both TtFAR and TtAT activities, we developed in vitro assays with microsomes prepared from the cmy228 yeast strain and from the engineered yeast (gat1⌬ gat2⌬ ϩ (pADH::FARAT(LEU2)), hereafter referred to as cmyFARAT). In particular, it remained to be determined whether the TtAT domain displays GPAT and/or DHAPAT activity. In the presence of [ 14 C]G 3 P, yeast microsomes from cmy228 led to the synthesis of radiolabeled lysophosphatidic acid (LPA), confirming the GPAT activity of GAT1 as well as of phosphatidic acid, monoacylglycerol, and diacylglycerol because of endogenous microsomal yeast activities (Fig. 4A). Under the same conditions but with microsomes from cmyFARAT, only LPA and phosphatidic acid could be detected, and the specific activity was more than 12 times lower (3.9 and 0.3 nmol/mg/min for GAT1 and TtFARAT, respectively, Fig. 4A). In addition, when unlabeled DHAP was added to the reaction mixture as a competitive substrate, the acyltransferase activity of GAT1 remained unaffected, whereas that of TtFARAT was 79% reduced (Fig. 4B).
In the presence of [ 14 C]DHAP, the synthesis of radiolabeled acyl-DHAP as well as traces of most probably acyl-dihydroxyacetone were observed with both microsomal preparations, but microsomes from cmyFARAT displayed a specific activity that was more than twice higher (3.2 and 1.5 nmol/mg/min for TtFARAT and GAT1, respectively, Fig. 4C). In the presence of unlabeled G 3 P as a competitive substrate, the acyltransferase activity of GAT1 was 85% reduced, whereas that of TtFARAT was unaffected (Fig. 4D). Together, these in vitro studies confirmed that GAT1 is a preferentially a GPAT but also displays significant DHAPAT activity, whereas TtFARAT is primarily a DHAPAT because it is 10 times more active with DHAP than with G 3 P.
Palmitoyl-CoA Is the Preferred Substrate of Both Activities Carried by TtFARAT-In vitro assays with yeast microsomes were further used to study the acyl chain specificity of the TtFAR and TtAT domains. Concerning the FAR domain of TtFARAT, in vitro assays in the presence of [ 14 C]16:0-CoA as a substrate showed that only the presence of NADPH allowed the detection of fatty alcohol (Fig. 5A) and confirmed that TtFAR is Reactions were stopped by adding 600 l of 1% HClO 4 . Lipids were extracted, and half of the organic phase was analyzed by thin-layer chromatography, whereas specific activities were calculated by quantifying the other half using scintillation spectrometry. a fatty acyl-CoA reductase. Assays in the presence of different radiolabeled acyl-CoAs indicated that the FAR specific activity of TtFARAT was about 3.5 and 16 times higher with 16:0-CoA than with 18:0-and 18:1-CoA, respectively (Fig. 5B). In comparison with the results obtained in vivo (Fig. 2), these assays stressed the preference of the reductase activity of TtFARAT for 16:0-CoA. When the DHAPAT activity of TtFARAT was tested using different acyl-CoA as acyl donors, 16:0-CoA also appeared by far as the preferred substrate because all other acyl-CoA tested resulted in 85-95% lower activities (Fig. 5C).

Reconstitution of Ether Lipid Biosynthesis in Yeast-Subcellular localization experiments showed that
TtFARAT is localized in the peroxisomes, whereas in vivo characterization and in vitro assays indicated that it carries both FAR and DHAPAT activities, strongly suggesting that the TtFARAT protein could be involved in ether lipid biosynthesis. Therefore, we tried to reconstitute ether lipid biosynthesis in yeast by expressing the ADPS from T. thermophila (TTHERM_00053800, GenBank TM accession number XM_001007509.2) in cmyFARAT. ADPS catalyzes the formation of the ether bond by exchanging the acyl chain of sn-1-acyl-dihydroxyacetone phosphate with a fatty alcohol. As shown in Fig. 6A, coexpression of both proteins resulted in the appearance of two new compounds that fragmentation identified as hexadecylglycerol-bistrimethylsilyl ( Fig. 6B) and octadecylglycerol-bistrimethylsilyl. The presence of these compounds in total fatty acyl chains demonstrated that coexpression of TtFARAT and TtAGPS successfully reconstituted ether lipid biosynthesis in yeast. Nevertheless, acyl chains and alcohols represented, after 2 days of expression, about 59 and 40% of the total, respectively, whereas alkyls were only minor components (less than 1% of the total).
To determine in which lipids the ether bond was present, lipid extracts were prepared from yeasts coexpressing TtFARAT and TtAGPS and fractionated to isolate the major glycerolipid subclasses. Solid phase separation of neutral and polar lipids indicated that if acyl chains were about equally distributed in both fractions, most of the alcohols were found in the neutral lipid fraction, whereas about 85% of the alkyl chains were associated with the polar lipids (Fig. 6C). Among the different phospholipids, phosphatidylinositol and phosphatidylserine subclasses were particularly enriched in ether bonds (Fig.  6D). The presence of several sn-1-alkyl-glycerolipids in the resulting transgenic yeast suggests that yeast endogenous activities involved in glycerolipid biosynthesis did not discriminate against the presence of an ether bond at the sn-1 position of the glycerol backbone.

DISCUSSION
Overall, this study demonstrates, by in vivo and in vitro approaches, that TtFARAT is a bifunctional protein involved in ether lipid biosynthesis. The N-terminal part was functionally characterized as a fatty acyl-CoA reductase, whereas the C-terminal domain is a DHAPAT activity. The fact that similar results were obtained when expressing the complete TtFARAT protein or each domain individually suggests that each activity is present in an autonomously folding domain. These results indeed confirmed a bioinformatics search for fused genes in the genome of T. thermophila that was conducted during the course of this study (28). This in silico approach predicted that TTHERM_00221020 may code for a protein with both FAR and DHAP activities, leading to an updated annotation in the T. thermophila genome database, where TtFARAT is currently named ART1 for acyl-CoA reductase/transferase (28). Such a fusion gene is restricted to few other ciliates, such as Paramecium tetraurelia, several amebozoans including Dictyostelium discoideum, and the oomycete Phytophthora infestans, suggesting that the same gene fusion event occurred independently in several distant phylogenetic groups (29).
Detection of several sn-1-alkyl-glycerolipids in the yeast expressing TtADPS and TtFARAT suggests that 1-alkyl-DHAP was exported from the peroxisomes to the endoplasmic reticulum, where it was reduced to LPA, acylated at the sn-2 position to form phosphatidic acid, and then further converted to various sn-1-alkyl-glycerolipids. These results indicate that yeast endogenous activities involved in phospholipids biosynthesis, therefore, did not discriminate against the presence of an ether bond at the sn-1 position of the glycerol backbone. In contrast, the lower abundance of ether bonds in the neutral lipid fraction suggests that yeast enzymes involved in triacylglycerol biosynthesis may discriminate against such lipids. Because yeast is normally devoid of fatty alcohols and ether lipids, our results also suggest that coexpression of TtFARAT and TtAGPS is nec- The cmyFARAT yeast strain was grown for 24 h at 30°C. Microsomes were prepared, and enzyme activities were assayed as indicated under "Experimental Procedures." Reactions were stopped by adding 600 l of 1% HClO 4 , and were lipids extracted. For FAR activity, radiolabeled fatty alcohols were identified by comigration with unlabeled standards and quantified by autoradiography. For DHAPAT activity, radiolabeled products present in the organic phase were quantified by scintillation spectrometry. and heptadecanoic acid (C17:0) were used as internal standards (IS). The peaks indicated by the arrow and the asterisk correspond to hexadecylglycerol-bistrimethylsilyl and octadecylglycerol-bistrimethylsilyl, respectively. Yeast cultures were grown for 5 days at 30°C. Lipids were transmethylated, and free hydroxyl groups were derivatized to trimethylsilyl ethers before separation by GC and detection by mass spectrometry. B, mass spectra and drawing representing fragmentation of hexadecylglycerol-bistrimethylsilyl (TMS). MW, molecular weight. C, partition of fatty acyls, alkyls, and alcohols in the neutral and polar lipid fractions. D, partition of fatty acyls and alkyls among the major phospholipid classes. Yeast cultures were grown for 2 days at 30°C, and lipids were extracted as indicated under "Experimental Procedures." Polar and neutral lipids were separated using solid phase extraction, whereas the major phospholipid classes were separated by thin-layer chromatography. The acyl chain composition was then analyzed by gas chromatography and detection by mass spectrometry. Partition is expressed as percent of total acyls, alkyls, or alcohols with standard deviation (n ϭ 4). PI, phosphatidylinositol; PS, for phosphatidylserine; PC, for phosphatidylcholine; PE, phosphatidylethanolimane. essary and sufficient to produce ether lipids in yeast. Finally, the strong predominance of 1-O-hexadecyl-ether lipids suggest that TtADPS is rather specific for 16-OH. Because our in vitro assays also stressed the clear preference of both activities carried out by TtFARAT for 16-carbon atom substrates, the three enzymes initiating ether lipid biosynthesis have apparently strong chain length specificities. The latter are most probably at the origin of the presence of predominantly 1-O-hexadecylether lipids in T. thermophila (14).
According to our biochemical characterization, TtFARAT produces the 16:0-fatty alcohol and sn-1-acyl-dihydroxyacetone phosphate substrates required by TtADPS to generate the ether bond and, thereby, initiates ether lipid biosynthesis in the peroxisomes (Fig. 7). In mammals, sucrose density gradient centrifugation, cross-linking, and coimmunoprecipitation studies have shown that DHAPAT and ADPS form a 210-kDa protein complex located on the luminal side of the peroxisomal membranes (30,31). In addition, radiation inactivation experiments showed, in guinea pig liver, that DHAPAT and ADPS had native molecular sizes of 62 and 79 kDa, respectively, and that, in situ, active functional units were monomers (32). In organisms having FAR-DHAPAT fusion genes, like T. thermophila, the FAR activity would also be part of such a complex. Because an antibody against the human FAR protein is now available (33), again performing such cross-linking and coimmunoprecipitation experiments could indicate whether DHAPAT and ADPS also form a trimeric complex with FAR in mammals. The TtFARAT bifunctional protein also raises questions about the localization of these activities on the peroxisomal membrane. DHAPAT and ADPS have been shown to be located inside the peroxisomes because their activities were resistant to trypsin treatment (34,35). Crystallographic structure studies suggest that ADPS membrane binding is due to two helices containing several basic amino acids creating an electropositive surface for interaction with phospholipids (36). Because in silico analysis of human DHAPAT with sequence analysis algorithms does not reveal any transmembrane segments with high reliability, this protein might also be associated with the luminal side of the peroxisomal membrane by hydrophobic and/or electrostatic interactions. Regarding TtFARAT, the presence of a type 1 peroxisomal targeting signal (PST1) also suggests luminal localization (37). In such a model, TtFARAT and TtAGPS would interact on the luminal side of the peroxisomal membrane, forming a catalytic complex generating 1-alkyl-DHAP from palmitoyl-CoA and DHAP. Nevertheless, this model does not fit with the recent study from Honsho and co-workers (13) suggesting that FAR1 is associated with the peroxisomes via two transmembrane domains present in its C terminus, whereas its large N-terminal catalytic domain is located on the cytosolic side. This topology fits with a previous study showing that FAR activity was sensitive to trypsin even without detergent disruption of the peroxisomal membranes (38). Although the situation may be different in organisms having TtFARAT-type bifunctional proteins, a physical separation may be necessary because both FAR and DHAPAT enzymatic activities use palmitoyl-CoA as a substrate. On the other hand, no transmembrane domain between the FAR and AT domains was detected by the sequence analysis programs we used. In addition, the presence of FAR activity outside of the peroxisomes rather complicates the ether biosynthetic pathway because the next step catalyzed by ADPS resides inside.
The term bifunctional is commonly used for two different types of proteins. In the first case, because of plasticity in substrate specificity (promiscuous enzymes) and/or catalysis (prolific enzymes), the enzyme activity results in different products. An example of this class is the most abundant protein on earth, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which catalyzes the fixation of CO 2 (carboxylation) and O 2 (oxygenation) on ribulose-1,5-bisphosphate in photosynthesis and photorespiration, respectively (39). Such proteins carry similar reactions in a single active site that accepts various substrates and/or different conformations for dual reactions. Other examples of such bifunctional proteins include bifunctional desaturase inserting the double bond at positions n-3 and n-6 (40), bifunctional wax ester synthase/diacylglycerol acyltransferase accepting both fatty alcohols and diacylglycerol as acyl acceptors (5), and bifunctional glucosyltransferase conjugating glucose with both amine (R-NH 2 ) and hydroxyl (R-OH) groups (41). In the second case, the enzyme catalyzes two separate reactions at two different active sites. One well known example is the peroxisomal D-bifunctional protein that catalyzes ␤-oxidation steps two (enoyl-CoA hydratase) and three (hydroxyacyl-CoA dehydrogenase) (42). Examples of this type occur much less frequently in eukaryotes because such bifunctional proteins usually result from gene fusion events, therefore requiring multiple active domains, each having independent history and function. In such an event, the regulatory elements of the second gene are usually lost so that expression of the resulting fusion gene only depends on the original promoter of the first gene. This is why gene fusion almost only occurs between genes belonging to the same metabolic pathway. In addition, such an event needs to confer a selective advantage to be retained by the following generations. A gene fusion between two successive steps of the same metabolic pathway is the most common, but fusions between non-consecutive steps have also been retained, for example the bifunctional aspartate kinase of plants that catalyzes the first and the third steps of methionine and threonine biosynthesis (43). TtFARAT represents a novel bifunctional protein in that it instead catalyzes two parallel reactions (FAR and DHAPAT), providing both substrates for a third reaction (ADPS).