Characterization of 2-Enoyl Thioester Reductase from Mammals AN ORTHOLOG OF Ybr026p/Mrf1 (cid:1) p OF THE YEAST MITOCHONDRIAL FATTY ACID SYNTHESIS TYPE II*

A data base search with YBR026c/MRF1 (cid:1) , which en-codes trans -2-enoyl thioester reductase of the intramitochondrial fatty acid synthesis (FAS) type II in yeast (Torkko, Schmitz, A. J., Airenne, T. T., Gur-vitz, A., and Hiltunen, K. J. (2001) Mol. Cell. Biol. 21, 6243–6253), revealed the clone AA393871 ( Hs Nrbf-1, nuclear receptor binding factor 1) in human EST data bank. Expression of Hs Nrbf-1, tagged C-terminally with green fluorescent protein, in HeLa cells, resulted in a punctated fluorescence signal, superimposable with the MitoTracker Red dye. Wild-type polypeptide was immunoisolated from the extract of bovine heart mitochondria. Recombinant Hs Nrbf-1p reduces trans -2-enoyl-CoA to acyl-CoA with chain length from C6 to C16 in an NADPH-dependent manner with preference to medium chain length substrate. Furthermore, expression of HsNRBF-1 in the ybr026c (cid:1) yeast strain restored mitochondrial respiratory function allowing growth on glycerol. These findings provide evidence that Nrbf-1ps act as a mitochondrial 2-enoyl thioester reductase, and mammalian cells may possess bacterial type fatty acid synthetase (FAS type II) in mitochondria, in addition to FAS type I in the cytoplasm. trifluoroacetic acid, and subjected to MALDI-TOF MS. Transmission EM of ybr026c (cid:2) Cells Expressing HsNrbf-1p— For the electron microscopy studies S. cerevisiae ybr026c (cid:2) cells overexpressing Hs Nrbf-1p or tr Hs Nrbf-1p were transferred from overnight cultures in synthetic medium lacking uracil and containing 2% (w/v) D -glucose (25) to oleic acid medium (26) and grown for 17 h at 30 °C. BJ 1991 wild-type cells and ybr026c (cid:2) cells were grown overnight on 1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) D -glucose and then transferred to oleic acid medium. The cells were fixed in 2.5% glutaraldehyde in 100 m M sodium phosphate buffer, pH 7.4, pelleted, and immersed in 2% agar (w/v) in PBS. Yeast cells in agar blocks were further post-fixed in 1% osmium tetroxide (w/v), dehydrated in acetone, and embedded in Em-bed 812 (Electron Microscopy Sciences, Fort Washington, PA). Thin sections of 80 nm were cut with a Reichert Ultracut ultramicrotome and examined using a Philips CM100 transmission electron microscope. Others— The generation of the S. cerevisiae BJ1991 ybr026c (cid:2) strain used in the transformations with pYE352:: HsNRBF-1 or pYE352:: trHsNRBF-1 has been described previously (27). Transformed yeast strains were maintained on synthetic medium lacking uracil (SC-U) and supplemented with 2% (w/v) D -glucose and tested for complementation on SC-U containing 3% (w/v) glycerol as a nonfer- mentable carbon source. Antibody against human Nrbf-1 was raised by immunizing rabbits with purified recombinant Hs Nrbf-1p. The IgG fraction from the antisera was purified by using the Econo-Pac Serum IgG purification kit (Bio-Rad). mammalian ortholog of S. cerevisiae Ybr026p/ Mrf1 (cid:1) p and C. tropicalis Etr1p. (i) Specifically, multiple amino acid sequence alignment shows that human, bovine, and rat proteins are sequence-related to the yeast enzymes and belong to the medium chain alcohol dehydrogenase/reductase super-family. (ii) Expression of the pEGFP-N1:: HsNRBF-1 in cultured HeLa cells resulted in cellular punctated fluorescent pattern, which superimposed with the signal from mitochondria stained with MitoTracker Red dye. This fluorescence pattern points to mitochondrial localization of Hs NRBF-1p, similar to that of Ybr026p and Etr1p in yeast. Immunoblotting of subcellular fractions of rat heart showed that anti-HsETRAb recognizes a polypeptide corresponding to the predicted size of the reductase subunit in the mitochondrial fraction. (iii) Het-erogeneous expression of HsNRBF-1 in the ybr026c (cid:2) strain restored growth of the yeast cells on glycerol, a function that requires a competent mitochondrial respiratory chain, defective in the ybr026c (cid:2) cells. Also the development of enlarged mitochondria was dependent on the presence of the mitochondrial targeting signal in Hs Nrbf-1p. (iv) Purified recombinant Nrbf-1ps are enzymatically active, catalyzing the NADPH-de-pendent reduction of trans -2-enoyl-CoA to acyl-CoA, a reaction also catalyzed by Ybr026p and Etr1p. (v) Finally, Bt Nrbf-1p can be purified from bovine heart.

Fatty acid synthesis (FAS) 1 can apparently take place in various subcellular organelles in eukaryotes. The eukaryotic extracytosolic FASs, like the fungal mitochondrial (1,2), plant mitochondrial (3), or plastid (4) systems, are similar to the prokaryotic type of FAS (type II) in terms of having a set of separate monofunctional enzymes (5) in contrast to the eukaryotic cytosolic FAS (type I), which consists of multifunctional polypeptide(s) (6,7). Fatty acid chain elongation in the mammalian and fungal endoplasmic reticulum also resembles FAS type II, except for the facts that it is membrane-associated (8) and has CoA as an acyl group carrier instead of acyl carrier protein (ACP).
The properties of the eukaryotic FASs and their role in metabolism appear to vary depending on the organism and the subcellular organelle; this is analogous to the ␤-oxidation of fatty acids. Although both mitochondrial and peroxisomal ␤-oxidation perform chain shortening of acyl-CoA substrates, the kinetic properties of the enzymes differ, and they clearly do not represent a metabolic redundancy, as exemplified by inborn errors of fatty acid oxidation (for review see Ref. 9). The disruption of any of the mitochondrial FAS type II genes in yeast results in a respiration-deficient phenotype (1,2,10), although the cytosolic FAS is still operational. Also the mitochondrial FAS in the yeast cannot compensate for a defective cytosolic pathway, and the inactivation of the cytosolic FAS is lethal, unless the cells are supplemented with fatty acids in the culture media (11).
We have recently identified a novel type of 2-enoyl thioester reductase from Candida tropicalis (Etr1p) and Saccharomyces cerevisiae (Ybr026p) that is involved in mitochondrial FAS (12). The ybr026c⌬ strain exhibits reduced mitochondrial cytochrome contents and an inability to grow on nonfermentable carbon sources (13). The yeast FAS type II 2-enoyl thioester reductases are homodimeric, NADPH-dependent, and are not sequence-related to the homotetrameric reductases of bacterial FAS type II.
Rat nuclear receptor binding factor-1 (RnNrbf-1p), which was described as homolog of the Ybr026p/Mrf1Јp by protein data base searches, was observed previously to interact with peroxisome proliferator-activated receptor ␣ as well as with a number of other nuclear receptors in the yeast two-hybrid assay (14). In the current work open reading frames (ORF) encoding the human homolog (HsNrbf-1p) of the rat Nrbf-1p was identified in human EST data bank, and the cDNA encoding the bovine homolog (BtNrbf-1p) was cloned. The data demonstrated that Nrbf-1ps are mitochondrial proteins that catalyze an NADPH-dependent reduction of trans-2-enoyl thioesters to the corresponding saturated acyl thioesters. As the expression of human NRBF-1 in the ybr026c⌬ yeast strain rescues the respiration-deficient phenotype, it is concluded that Nrbf-1ps are the orthologs of the yeast 2-enoyl thioester reductases, enzymes previously unidentified in mammals.

EXPERIMENTAL PROCEDURES
Isolation of Human NRBF-1 cDNA and Northern Blot Analysis-The S. cerevisiae gene YBR026c was used to search for human EST clones in the expressed sequence tag data base at the National Center for Biotechnology Information. An EST clone, AA393871 (IMAGE ID 504485), was obtained from I.M.A.G.E. Consortium (UK Human Genome Mapping Project Resource Centre, Hinxton, Cambridge, UK) and se-* This work was supported in part by grants from the Academy of Finland and Sigrid Jusélius Foundation. 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AY256973.
ʈ quenced. The ORF of the AA393871 (HsNRBF-1) clone was amplified with Pfu polymerase (Stratagene, La Jolla, CA) using the primer pair HsNRBF-1comp5Ј (5Ј-GAG CTC TAG AAG ATG TGG GTC TGC AGT  ACC C-3Ј) and HsNRBF-1comp3Ј (5Ј-CG AGC TCG AGT TCA CAT  GGT GAG AAT CTG CTT-3Ј), which introduced XbaI and XhoI sites (underlined) to the ends of the predicted ORF. The nucleotide triplets encoding an initiating starting methionine and stop codon are indicated in the oligonucleotides with boldface letters. The resulting DNA fragment was subcloned into pBluescript SK(ϩ) (pSK) vector (Stratagene). cDNA was removed from the pSK vector by digestion with XbaI and XhoI and ligated into similarly digested pYE352 behind the S. cerevisiae catalase A (CTA1) promoter (15), resulting in pYE352::HsNRBF-1. For Northern blot analysis, a human Multiple Tissue Northern Blot TM (Clontech, Palo Alto, CA) containing poly(A ϩ ) RNA from various human tissues was hybridized with 32 P-labeled HsNRBF-1 cDNA. Human ␤-actin cDNA (supplied with the kit) was used as a control probe. Hybridization was done in ExpressHyb TM (Clontech) solution according to the manufacturer's instructions, and the hybridized fragments were visualized by autoradiography at Ϫ70°C for 24 h using X-Omat TM AR imaging film (Eastman Kodak Co.).
Cloning of the NRBF-1 cDNA from Bovine Heart-The total RNA was isolated from bovine heart tissue using QuickPrep system (Amersham Biosciences). cDNA was amplified from isolated RNA with RobustRT-PCR kit (Finnzymes, Espoo, Finland), which contains a high fidelity DyNAzyme EXT, using the primer pair BtNRBF1-5Ј (5Ј-TGG AGG GAA CAT GTG GG-3Ј) and BtNRBF1-3Ј (5Ј-ATC ACA T(G/A)G TGA GAA TCT GCT T-3Ј). The composition of the BtNRBF1-5Ј was based on the sequence of bovine EST clone BE682523, containing the 5Ј end of the cDNA obtained by the data base search with the full-length human NRBF-1 sequence. The composition of the BtNRBF1-3Ј was based on the consensus sequence obtained from the alignment of human, rat, and mouse sequences. The PCR product was ligated to pSTBlue vector using Acceptor Vector kit (Novagen, Madison, WI), and both strands of the insert were sequenced. The resulting plasmid was named pSTBlue::BtNRBF-1.
Overexpression and Purification of Recombinant Human and Bovine Nrbf-1p-To produce the recombinant, truncated variant of HsNrbf-1p, which was deleted for 31 N-terminal amino acid residues, based on the amino acid sequence comparison to the mature form of Mrf1-p (13), nucleotides 90 -1119 from AA393871 were amplified with primers HsNRBF1-PET3a-5Ј (5Ј-TT TTT TTT CAT ATG GCC TCC TCC TAC TCC GCA TCC-3Ј) and HsNRBF1-PET3a-3Ј (5Ј-TTT TTT TTT CAT ATG TCA CAT GGT GAG AAT CTG CTT TGA AGA TAT G-3Ј), containing NdeI sites (underlined). After digestion with NdeI, the resulting PCR product was cloned into NdeI-digested pET3a vector (Novagen) to generate pET3a::trHsNRBF1. The bovine construct was generated by amplifying nucleotides 95-1124 from pSTBlue::BtNRBF1, deleting also 31 N-terminal residues, with primers BovETR5Ј (5Ј-TT TTT TTT CAT ATG TCC GCC TCC TTC TCT GCC-3Ј) and BovETR3Ј (5Ј-TTT GGA TCC TCA CAT GGT GAG AAT CTG CTT TGA AGA CAC-3Ј), which contained initiation and termination codons (boldface) within the coding sequence and NdeI (BovETR5Ј) and BamHI (BovETR3Ј) sites (underlined). The PCR product was digested with BamHI and NdeI and ligated into similarly digested pET3a vector to obtain pET3a::trBtNRBF1. After verification of the sequences and orientation of the inserts, the plasmid DNA was transformed into E. coli BL21 (DE3) pLysS cells and the expression of the recombinant protein was induced in M9ZB medium by the addition of isopropyl-␤-D-thiogalactoside to a final concentration of 1.0 mM according to the manufacturer's protocol. After incubation for an additional 10 h at 37°C, the cells were washed and stored at Ϫ20°C until used. Although the immunological cross-reaction of the antibodies raised against Etr1p from C. tropicalis (anti-Etr1pAb; Ref. 12) with mammalian homologs was weak, the antibody could be used for monitoring the purification of recombinant HsNrbf-1p; in the meantime an antibody to HsNrbf-1p (anti-HsETRAb) was raised and used to monitor the purification of recombinant BtNrbf-1p.
2-Enoyl Thioester Reductase Activity and Reaction Product Analysis-Enoyl reductase activity was measured using trans-2-hexenoyl-CoA (60 M), synthesized by the mixed anhydride method (16) as described previously (17). For end product analysis 3 g of recombinant trHsNrbf-1p was incubated in the presence of 60 M trans-2-hexenoyl-CoA and 300 M NADPH in 1 ml of 20 mM potassium phosphate, pH 7.8, at 22°C. Taken aliquots of 100 l were acidified with 10 l of 2 M HCl, and samples of 20 l from the aliquots were subjected to a reversed phase chromatography (Waters Novapak C18, 2 ϫ 150 mm, Waters 626 pump, Waters 996 photodiode array detector, Waters Millipore, Millford, MA). The column was eluted with an isocratic flow of 0.2 ml/min with buffers A (25 mM CH 3 COONH 4 , pH 5.5) and B (80% CH 3 CN and 25 mM CH 3 COONH 4 , pH 5.5) in 83:17 ratio. Metabolites in the eluted peaks were identified by matrix-assisted laser desorption ionizationtime of flight-mass spectroscopy (MALDI-TOF MS) (ABI Voyager-DETM STR Biospectrometry Workstation).
Determination of Kinetic Parameters for HsNrbf-1p-The kinetic constants for reductase activities were determined using substrate concentrations of 1.25-200 M for trans-2-hexenoyl-CoA, trans-2-decenoyl-CoA, and trans-2-hexadecenoyl-CoA. Otherwise the assay mixture contained 125 M NADPH and 100 g of bovine serum albumin in 50 mM potassium phosphate, pH 7.5, in a final volume of 1 ml at 22°C. The kinetic data were transferred to Lineweaver-Burk plots by using GraFit computer software (Sigma). The catalytic turnover numbers were obtained by dividing the maximum velocities with the enzyme (subunit) concentrations in the reaction mixtures.
Fluorescence Microscopy-Attached HeLa cells (CCL-2; American Type Culture Collection) were grown on glass coverslips in Dulbecco's modified Eagle's media (Invitrogen) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin (Invitrogen). The transfection of the HeLa cells was done using FuGENE 6 reagent (Roche Diagnostics), according to the manufacturer's protocol, and using 6 l of FuGENE 6 with 2 g of either pEGFP-N1::HsNRBF-1 or pEGFP-N1. After transfection the cells were grown for 36 h and treated with the mitochondrion-selective fluorescent dye MitoTracker Red CMXRos (Molecular Probes Europe BV, Leiden, Netherlands) according to the manufacturer's instructions. Cells were washed in phosphate-buffered saline (Invitrogen) and fixed with 4% paraformaldehyde in 14 mM sodium phosphate, pH 7.4, 150 mM NaCl (PBS) for 20 min at 22°C. To minimize the background fluorescence, the fixed cells were incubated in 50 mM NH 4 Cl/PBS for 10 min before treatment with 0.2% (v/v) Triton X-100/PBS for 10 min at 22°C. Nuclear DNA was counterstained with 1 g/ml 4Ј,6-diamidino-2Ј-phenylindole dihydrochloride (DAPI), and the mounted (IMMU-MOUNT; Thermo Shandon, Pittsburgh, PA) slides were examined using a fluorescence microscope (Olympus Optica, Hamburg, Germany). Images were recorded with a CCD camera and processed with the Jasc Software Paint Shop Pro.
Isolation of Rat Heart Mitochondria-Crude mitochondria fraction from rat heart was isolated as described (18). The bacterial protease (Nagarse) used was from Sigma. Mitochondrial pellet was suspended to a buffer containing 10 mM Tris-Cl, pH 7.8, 250 mM sucrose, 0.2 mM EDTA, giving a protein concentration of 23 mg/ml. An aliquot of mitochondrial fraction (46 mg of protein) was purified further by isopycnic density gradient centrifugation (19) on a self-generated Percoll (Sigma) gradient (20) with an initial Percoll concentration of 32% (v/v). Gradient was generated by ultracentrifugation at 100,000 ϫ g for 30 min in Beckman VTi50 rotor using slow acceleration/deceleration mode. Gradient was unloaded in 2-ml fractions from the bottom of the tube, and marker enzyme activities, cytochrome c oxidase (21), esterase (22), and lactate dehydrogenase (23) for mitochondria, microsomes, and cytosol, respectively, were measured.
Isolation of Wild-type BtNrbf-1p-The mitochondria were isolated from bovine heart, obtained from a local slaughterhouse as described earlier (24), and stored at Ϫ70°C until used. The soluble mitochondrial extract was prepared by disrupting 2 g of mitochondria by sonication in 20 ml of 50 mM potassium phosphate, pH 7.8, containing 70 mM NaCl at 0°C, followed by ultracentrifugation at 100,000 ϫ g for 1 h. A sample of supernatant containing 120 mg of protein was dialyzed against 20 mM potassium phosphate, pH 7.8, and applied to a 2Ј,5Ј-ADP-Sepharose 4B column (1.0 ϫ 7.0 cm) equilibrated with dialysis buffer at the flow rate of 0.5 ml/min. The bound proteins were eluted with a NaCl gradient from 0 to 0.4 M (10 ml) and from 0.4 to 2 M (10 ml). The steep part of the gradient was implemented to prevent the excess dilution of BtNrbf-1p, as detected by immunoblotting with anti-HsETRAb. The fractions containing BtNrbf-1p were pooled, dialyzed against PBS, and subjected to immunoisolation. A suspension of 100 l of protein A-coated Dynabeads (Dynal, Oslo, Norway) was washed three times with 100 mM sodium phosphate buffer, pH 8.2, and incubated for 1 h at room temperature with 10 l of anti-HsETRAb or preimmune serum. The antibody-loaded beads were washed three times with PBS containing 0.1% Tween 20 (PBST) and transferred using the PickPen magnetic particle transfer device (Bio-Nobile, Turku, Finland) to a 400-l aliquot of the dialyzed sample containing BtNrbf-1p (catalyzing the reduction of 60 M trans-2-decenoyl-CoA at the rate of 0.6 nmol/min). After incubation at 4°C with gentle shaking the beads were separated, washed three times with PBST, and transferred to 20 l of SDS sample buffer containing 50 mM DTT. After 30 min of incubation at 37°C, the beads were removed, and the sample was acetylamidated with 110 mM iodoacetamide for 1 h at 37°C. After electrophoresis, bands were excised, destained, and dried. The gel pieces were rehydrated with trypsin solution (20 g/ml porcine trypsin in 40 mM NH 4 CO 3 containing 10% (v/v) CH 3 CN and 0.5% (w/v) ␤-octyl-D-glucoside) and incubated overnight at 37°C. Digested peptides were recovered by extraction with four 20-l aliquots of 0.1% trifluoroacetic acid in water containing 5, 30, 50, and 70% CH 3 CN, respectively. Extracts were pooled, dried, and dissolved into 5 l of saturated ␣-cyano-4-hydroxycinnamic acid in 40% CH 3 CN, 0.1% trifluoroacetic acid, and subjected to MALDI-TOF MS.
Transmission EM of ybr026c⌬ Cells Expressing HsNrbf-1p-For the electron microscopy studies S. cerevisiae ybr026c⌬ cells overexpressing HsNrbf-1p or trHsNrbf-1p were transferred from overnight cultures in synthetic medium lacking uracil and containing 2% (w/v) D-glucose (25) to oleic acid medium (26) and grown for 17 h at 30°C. BJ 1991 wild-type cells and ybr026c⌬ cells were grown overnight on 1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) D-glucose and then transferred to oleic acid medium. The cells were fixed in 2.5% glutaraldehyde in 100 mM sodium phosphate buffer, pH 7.4, pelleted, and immersed in 2% agar (w/v) in PBS. Yeast cells in agar blocks were further post-fixed in 1% osmium tetroxide (w/v), dehydrated in acetone, and embedded in Embed 812 (Electron Microscopy Sciences, Fort Washington, PA). Thin sections of 80 nm were cut with a Reichert Ultracut ultramicrotome and examined using a Philips CM100 transmission electron microscope.
Others-The generation of the S. cerevisiae BJ1991 ybr026c⌬ strain used in the transformations with pYE352::HsNRBF-1 or pYE352::trHsNRBF-1 has been described previously (27). Transformed yeast strains were maintained on synthetic medium lacking uracil (SC-U) and supplemented with 2% (w/v) D-glucose and tested for complementation on SC-U containing 3% (w/v) glycerol as a nonfermentable carbon source. Antibody against human Nrbf-1 was raised by immunizing rabbits with purified recombinant HsNrbf-1p. The IgG fraction from the antisera was purified by using the Econo-Pac Serum IgG purification kit (Bio-Rad). Restriction enzymes were purchased from New England Biolabs (Hertfordshire, UK) or from Amersham Biosciences. The use of laboratory animals was approved by the committee on animal experimentation of the University of Oulu.

Data Base Search, Tissue Distribution of the Expression, and
Amino Acid Sequence Alignment-A BLAST search (28) of protein data bases with S. cerevisiae Ybr026p/Mrf1Јp or C. tropicalis Etr1p revealed homologs from various eukaryotes, including human (Fig. 1). The corresponding human protein EST, AA393871, was sequenced, and the ORF was termed HsNRBF-1. A genomic data base search showed that the human genome contains only a single copy of the HsNRBF-1 gene annotated as CGI-63/NRBF-1. The gene is located in section p22.3 of chromosome 1 spanning over 37 kb and contains 10 exons and 9 introns.
The expression of HsNRBF-1 was analyzed in different human tissues by Northern blot analysis. The strongest signals of 1.4 kb for HsNRBF-1 were observed in the samples from skeletal and heart muscles ( Fig. 2A). Weak signals of the same size were observed in the samples from brain, placenta, liver, kidney, and pancreas, but no signal was observed in the sample from lung. Hybridization with a ␤-actin probe was used as a control for the amount of mRNA on the nylon blot (Fig. 2B). Two mRNAs of ␤-actin were visualized in heart and skeletal muscle, namely a band of 2 kb size and a smaller one of 1.6 -1.8 kb size as observed previously (29,30).
Because the Northern blot analysis indicated that the NRBF-1 was expressed in heart muscle, this tissue was chosen as a material for characterization of the mammalian Nrbf-1p originating from a wild-type source. The BtNRBF-1 cloned from bovine heart was 1133 bp long and contained an open reading frame of 1122 nucleotides, encoding a polypeptide of 373 amino acid residues. The similarities between human and bovine Nrbf-1p sequences were 88 and 85% at the nucleotide and amino acid level, respectively.
The amino acid sequence identities between HsNrbf-1p (Bt-Nrbf-1p) and Ybr026p and Etr1p were 40 (35%) and 36% (38%), respectively, and 80% (83%) to Rattus norvegicus Nrbf-1p (14). An amino acid identity of 42% (44%) was obtained with the annotated Ybr026p homolog in Caenorhabditis elegans. Fig. 1 shows that the identical/similar amino acid residues in these proteins formed clusters, which were found along the length of the polypeptides excluding the extreme N and C termini. The tyrosine residue critical for catalysis in the yeast enzymes (31) is also conserved in the mammalian protein (Tyr-94 in HsNrbf-1p).
Overexpression and Purification of HsNrbf-1p and BtNrbf-1p-To characterize further the functions of HsNrbf-1p and BtNrbf-1p, their variants deleted for the predicted mitochondrial targeting signals were overexpressed from a pET3a vector in Escherichia coli and purified to apparent homogeneity as described under "Experimental Procedures," yielding from 6.0 to 8.8 mg of protein from 2 liters of the bacterial culture. SDS-PAGE analysis showed a single protein band of 37 kDa, which is in agreement with the mass of predicted polypeptides (37,052 Da and 36,973 Da) encoded by HsNRBF-1 and Bt-NRBF-1 in pET3a. Size exclusion chromatography using a Superdex 200 HR column gave an estimated native mass of 65 kDa (Fig. 3), indicating that both proteins are homodimers.
Enzymatic Properties of 2-Enoyl Thioester Reductase-The amino acid sequence similarity of HsNrbf-1p and BtNrbf-1p with C. tropicalis Etr1p and S. cerevisiae Ybr026p prompted us to test whether they have a similar enzymatic activity. Therefore, HsNrbf-1p was incubated in the presence of trans-2-hexenoyl-CoA and NADPH, and samples were taken from the reaction at various time points for HPLC analysis (Fig. 4, panel  1). The oxidation of NADPH was accompanied by the disappearance of trans-2-hexenoyl-CoA with a concomitant accumulation of a metabolite (Fig. 4, panel 2, A-C) having the same retention time as hexanoyl-CoA (Fig. 4, panel 3, A-C). The accumulating metabolite was 2 mass units larger than trans-2-hexenoyl-CoA (863.7 m/z) in MALDI-TOF MS analysis consistent with the end product being hexanoyl-CoA (865.7 m/z). The data show that HsNrbf-1p is a 2-enoyl thioester reductase, reducing trans-2-enoyl thioesters to their saturated counterparts. NADH could not replace NADPH in the assay.
The kinetic parameters for the HsNrbf-1p were determined using trans-2-hexenoyl-and trans-2-decenoyl-CoA as a substrates. The centrations were blotted. The maximum k cat of 0.2 s Ϫ1 was obtained at 6 M trans-hexadecenoyl-CoA. The detected low activity under tested conditions hampered the estimation of kinetic parameters. When the BtNrbf-1p was tested with trans-2-decenoyl-CoA, the k cat was 0.52 Ϯ 0.02 s Ϫ1 , the K m 4.5 Ϯ 0.6 M Ϫ1 , and the k cat /K m 1.15 ϫ 10 5 M Ϫ1 s Ϫ1 , values that are comparable with those obtained for HsNrbf-1p. Subcellular Localization of Mammalian and Rat Nrbf-1p-The N-terminal extensions of human, bovine, and rat Nrbf-1p are rich in hydrophobic and positively charged amino acid residues, contain hydroxylated residues, and lack negatively charged residues; these are features of a mitochondrial targeting signal (32). Subcellular localization prediction of HsNrbf-1p, BtNrbf-1p, and rat Nrbf-1p gave 80 -97% probability that they are mitochondrial proteins (TargetP V1.0 (33) and Mito-ProtII 1.0a4 (34)). This prediction challenges the previous suggestion that rat Nrbf-1 is a cytosolic/nuclear protein. To reinvestigate the subcellular localization of mammalian Nrbf-1p, pEGFP-N1::HsNRBF-1 was expressed in HeLa cells (Fig. 5, A-C); the mitochondria were stained with MitoTracker Red (Fig. 5, C and G), and the nuclear DNA was counterstained with DAPI (Fig. 5, B and F). The cells transfected with pEGFP-N1::HsNRBF-1 show a well defined punctated fluorescence pattern (Fig. 5A) that superimposes with MitoTracker staining (Fig. 5D). In contrast, a diffuse uniform cellular fluorescent signal, not superimposable with MitoTracker staining, was observed in the cells transfected with control plasmid (pEGFP-N1) (Fig. 5, E and H). Separated experiments were carried out with HeLa cells expressing the GFP attached Nterminally to HsNrbf-1p and the outcome was diffuse fluorescent signal, similar to that obtained with control plasmid (data not shown). The localization of wild-type Nrbf-1p was also studied with subcellular fractionation of rat heart organelles by differential and isopycnic centrifugations in self-generated Percoll gradient. Cytochrome c oxidase was concentrated at the density of 1.08 -1.1 g/ml (Fig. 5I). Immunoblotting of the Percoll gradient fractions with anti-HsETRAb (Fig. 5I) visualized, in the fractions containing cytochrome c oxidase activity, a 37-kDa band that corresponds to the polypeptide size predicted from NRBF-1_Rn (Fig. 1). Because 2-enoyl thioester reductase activity was detected in the same fractions, the findings suggested mitochondrial localization of Nrbf-1p also in rat heart.
Purification of Nrbf-1 from Bovine Heart-The BtNrbf-1p from the soluble extract of isolated bovine heart mitochondria, partially purified with 2Ј,5Ј-ADP-Sepharose affinity column, was subjected to immunoaffinity purification using anti-HsE-trAb bound to protein A-coated magnetic beads as described under "Experimental Procedures." The reductase activity in the sample decreased with anti-HsETRAb in a dose-dependent manner but not in the control experiments carried out with the beads treated with preimmune serum (Fig. 6A). Analysis of immunoisolated proteins by SDS-PAGE revealed a band of 37 kDa (Fig. 6B) that was excised and in-gel digested. With the peptide fingerprint measured by MALDI-TOF MS (Fig. 6C), the FIG. 4. The measurement of 2-enoyl thioester reductase activity and the analysis of the reaction products. Panel 1, HsNrbf-1p was incubated with NADPH and trans-2-hexenoyl-CoA as described under "Experimental Procedures." The initial absorbance was set at zero, and reaction was monitored by measuring the oxidation of NADPH to NADP ϩ at 340 nm. trans-2-Hexenoyl-CoA (60 M) was added to the mixture at t 0 , and aliquots of 100 l were taken at 0 (t 0 ), 7 (t 1 ), and 60 min (t 2 ) for HPLC analysis. corresponding protein was unambiguously identified as Bt-Nrbf-1p. The peptides detected covered a large part of the sequence between amino acids 45 and 325. N-terminal peptides were not found, in agreement with mitochondrial targeting signal cleavage required for mitochondrial import.
Physiological Function of HsNrbf-1p Complementation Studies with Yeast-In order to study whether HsNrbf-1p is the human ortholog of the yeast Ybr026p and Etr1p, ybr026⌬ cells were transformed with pYE352::HsNBRF-1 or pYE352::trHsNBRF-1, encoding HsNrbf-1p and trHsNrbf-1p, respectively, and tested for growth on glycerol. The ybr026c⌬ strain and ybr026c⌬ expressing catalase A were unable to grow on glycerol. Expression of HsNRBF-1 was able to rescue the growth of ybr026⌬ cells (Fig. 7) similar to YBR026c (12). Growth on glycerol was not rescued by expression of the trHsNrbf-1, a variant of the human protein truncated for mitochondrial targeting signal (Fig. 7), although it was expressed as indicated by immunoblotting with anti-Etr1pAb (data not shown).
When transmission electron microscopy was carried out, rudimentary mitochondria were visualized in ybr026c⌬ cells as well as in the ybr026c⌬ expressing trHsNrbf-1p (Fig. 8, B and C). Compared with mitochondria in wild-type cells (Fig. 8A), enlarged mitochondria were developed upon HsNRBF-1 expression (Fig. 8D). These data are similar to that obtained by expression of yeast 2-enoyl thioester reductase in ybr02c6⌬ cells with or without N-terminal mitochondrial targeting signal (12). DISCUSSION Although mammalian mitochondria are known to have 2-enoyl thioester reductase activity (35), the enzyme involved in this process has not been identified previously, and its role in metabolism remains open. This work provides evidence that Nrbf-1p is the mammalian ortholog of S. cerevisiae Ybr026p/ Mrf1Јp and C. tropicalis Etr1p. (i) Specifically, multiple amino acid sequence alignment shows that human, bovine, and rat proteins are sequence-related to the yeast enzymes and belong to the medium chain alcohol dehydrogenase/reductase superfamily. (ii) Expression of the pEGFP-N1::HsNRBF-1 in cultured HeLa cells resulted in cellular punctated fluorescent pattern, which superimposed with the signal from mitochondria stained with MitoTracker Red dye. This fluorescence pattern points to mitochondrial localization of HsNRBF-1p, similar to that of Ybr026p and Etr1p in yeast. Immunoblotting of subcellular fractions of rat heart showed that anti-HsETRAb recognizes a polypeptide corresponding to the predicted size of the reductase subunit in the mitochondrial fraction. (iii) Heterogeneous expression of HsNRBF-1 in the ybr026c⌬ strain restored growth of the yeast cells on glycerol, a function that requires a competent mitochondrial respiratory chain, defective in the ybr026c⌬ cells. Also the development of enlarged mitochondria was dependent on the presence of the mitochondrial targeting signal in HsNrbf-1p. (iv) Purified recombinant Nrbf-1ps are enzymatically active, catalyzing the NADPH-dependent reduction of trans-2-enoyl-CoA to acyl-CoA, a reaction also catalyzed by Ybr026p and Etr1p. (v) Finally, BtNrbf-1p can be purified from bovine heart.
From the evolutionary point of view, HsNrbf-1p and its orthologs in other eukaryotes are unique among monofunctional, non-flavin-containing 2-enoyl thioester reductases as they are members of the medium chain alcohol dehydrogenase/reductase superfamily, whereas other known reductases belong to the short chain alcohol dehydrogenase/reductase family. The others include, as prototypes of the family, triclosan-sensitive FabI from E. coli and isoniazid-sensitive InhA from Mycobacterium tuberculosis (36,37). TSC13/YDL015c in S. cerevisiae has also been identified to encode a 2-enoyl reductase, which catalyzes the last reaction of each cycle of fatty acid chain elongation in the endoplasmic reticulum (38). A novel NADPHdependent 2-enoyl-CoA reductase from the short chain alcohol FIG. 5. Determination of the subcellular localization of HsNrbf-1p by fluorescence microscopic analysis and subcellular fractionation. HeLa cells (ATCC CCL-2) cultured on glass coverslips were transfected with either pEGFP-N1::HsNBRF-1 (A) or pEGFP-N1 plasmid (E). The cells were treated with MitoTracker Red CMXRos (50 -100 nM), identified by the color red (in C and G). The cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 after which the nuclear DNA was counterstained with DAPI, identified by the blue color (in B and F). The slides were examined by fluorescent microscopy to verify the expression of GFP fusion protein (in A and E). The green fluorescence (A) of the cells transfected with pEGFP-N1::HsNBRF-1 is specific for the mitochondria as indicated by co-localization with MitoTracker (D). In the cells transfected with pEGFP-N1 the fluorescence from GFP is observed in the cytoplasm and nucleus (E). D, a composite image of A-C. H, a composite image of E-G. I, crude mitochondrial preparation of rat heart was fractionated by isopycnic ultracentrifugation on a self-generated Percoll gradient. The columns show the distribution of cytochrome c oxidase (black), esterase (gray), and lactate dehydrogenase (white) activities in the gradient. The solid line indicates the density of the fractions. Immunoblotting of the fractions with anti-HsETRAb, which visualized a band at 37 kDa, is also shown. dehydrogenase/reductase family has been characterized recently from human and mouse peroxisomes, and it has been hypothesized to contribute to peroxisomal fatty acid chain elon-gation (39). Both peroxisomal and mitochondrial 2,4-dienoyl-CoA reductases, which catalyze the NADPH-dependent reduction of 2,4-dienoyl-CoA to trans-3-enoyl-CoA and are auxiliary enzymes of ␤-oxidation of (poly)unsaturated fatty acids, are also short chain alcohol dehydrogenase/reductase proteins (40,41). Recently, Heath and Rock (42) identified as a third type of 2-enoyl thioester reductase from Streptococcus pneumoniae an NADH-dependent flavin enzyme FabK that has no similarity to FabI at the amino acid sequence level and is resistant to triclosan.
Mitochondrial targeting of HsNrbf-1-GFPp in cultured HeLa cells, immunological localization of Nrbf-1p in subcellular fractions of rat heart, and heterologously expressed HsNRBF-1 in yeast disagree with earlier reports (14) on cytosolic/nuclear localization and coactivator hypothesis of mammalian Nrbf-1p. This disagreement can be explained in terms of differences in experimental conditions, because the previous studies were carried out using rat Nrbf-1 with N-terminally attached hemagglutinin (HANrbf-1). In this work the expression of the wild-type HsNrbf-1p or C-terminally tagged HsNrbf-1p leaves the potential N-terminal mitochondrial targeting signal functioning. In addition to the cytosolic/nuclear localization of HANrbf-1 in HeLa cells, the coactivator hypothesis is based on the findings that rat Nrbf-1 interacts with peroxisome proliferator-activated receptor ␣ and other nuclear receptors, in the presence of respective ligands, in the yeast two-hybrid assays (14). However, the mitochondrial localization observed in this study necessitates a re-evaluation of the physiological significance of these interactions and the suggestion that the Nrbf-1p acts as a nuclear receptor coactivator.
The most significant function of mitochondrial FAS in fungi (2, 43) and in plants (3) is regarded as the generation of octanoyl-ACP for lipoic acid synthesis by lipoic acid synthase. Although a dietary supply of lipoic acid is crucial for the well FIG. 6. Immunopurification of native BtNrbf-1p. A, an aliquot of the pooled fraction containing bovine reductase from ADP-Sepharose column was treated with anti-HsETRAb and immobilized on protein A-coated magnetic beads. The initial reductase activity in the aliquot was set to 100%. The solid line indicates the remaining reductase activity after consecutive immunoaffinity isolation. The dashed line shows the control experiments with preimmune serum. B, silverstained SDS-PAGE of proteins eluted from the beads. The arrows on the left indicate the molecular size markers. The arrow on the right shows the band excised for mass analysis. The heavily stained bands at 50 and 23 kDa present heavy and light chain polypeptides of IgG, respectively. C, mass spectrum of the peptide fingerprint obtained after trypsin digestion of the excised band. The arrows indicate the peaks that match within 20 ppm to predicted tryptic digestion of BtNrbf-1p.