A very brief description of the procedure for generating BnDGAT1_(1–116)His₆ was presented previously in a conference proceeding document 23. A hydropathy plot 24 of the deduced amino acid sequence of BnDGAT1 13 indicated that the N-terminal region of the enzyme was relatively hydrophilic compared to the rest of the protein. Based on this information, we designed a construct which would result in the production of a gene product consisting of the first 116 amino acid residues of BnDGAT1 followed by a four amino acid residue (AALE) linker and 6 His residues. Automated DNA sequencing was performed across the cloning junctions and for the entire coding region, to verify there was no shift in the open reading frame. Induction of BnDGAT1_(1–116)His₆ synthesis by isopropyl β-D-thiogalactoside (IPTG) treatment of bacteria is shown in Figure 1A. Analysis of lysates from induced Escherichia coli (lanes 3 and 4) clearly showed the appearance of an abundant polypeptide with an apparent molecular mass of about 23 kDa, which was considerably larger than the calculated molecular mass of 13,278 Da for BnDGAT1_(1–116)His₆. The 23 kDa band was barely perceptible in the pellet protein (lane 5), indicating that the expressed gene product was soluble in the extraction buffer. The poly-His tag on BnDGAT1_(1–116)His₆ facilitated effective purification of the recombinant N-terminal fragment by immobilized nickel ion chromatography. Analysis of different elution fractions of the Ni²⁺-NTA-agarose column by SDS-PAGE is depicted in Figure 1B. The protein fraction that was eluted with 40 mM imidazole was highly purified based on SDS-PAGE (lane 4) and thus was used for subsequent characterization work. Analysis of purified BnDGAT1_(1–116)His₆ by MALDI-TOF mass spectrometry indicated a molecular mass of about 13.2 kDa, which was in close agreement with the calculated molecular mass (Figure 1C). Analysis of BnDGAT1_(1–116)His₆ by dynamic light scattering (DLS) indicated that the protein preparation was largely monodisperse and potentially suitable for crystallization. Small plate-like monoclinic crystals were grown from preparations of purified BnDGAT1_(1–116)His₆ (Figure 1D). The largest microcrystals, however, were too small (0.07 × 0.07 × 0.01 mm) for x-ray diffraction studies using a rotating anode x-ray source.

In a previous study, we demonstrated that [1-¹⁴C]oleoyl-CoA interacted with BnDGAT1_(1–116)His₆ using a low concentration range (0–5 μM) of thioester 23. In the current study, the range of thioester concentrations used in Lipidex-1000 binding assays was extended up to near 30 μM for [1-¹⁴C]oleoyl-CoA (Figure 2A). As well, binding studies were conducted with [1-¹⁴C]erucoyl-CoA for comparative purposes. Binding experiments with BSA, which is known to interact with acyl-CoAs 25, demonstrated that the Lipidex-1000 assay was effective and thus served as a positive control (data not shown). In contrast, we have previously shown that ovalbumin does not bind acyl-CoA using this assay 23. The binding of both acyl-CoA species to BnDGAT1_(1–116)His₆ occurred in a sigmoidal fashion. The binding ratio of thioester: BnDGAT1_(1–116)His₆ was based on the molecular mass of 13,278 Da calculated from the amino acid sequence of the N-terminal fragment. BnDGAT1_(1–116)His₆ bound more erucoyl-CoA than oleoyl-CoA at considerably lower concentrations of acyl-CoA indicating that the N-terminal fragment had a greater affinity for erucoyl-CoA than oleoyl-CoA. Analysis of the binding data using Scatchard plots 26 is depicted in Figures 2C and 2D. Both plots rose to a maximum and then decreased suggesting that the interaction between acyl-CoA and BnDGAT1_(1–116)His₆ involved positive cooperativity. Dissociation constants of about 17 μM and 2 μM were determined for the binding of oleoyl-CoA and erucoyl-CoA, respectively, to the high affinity state of BnDGAT1_(1–116)His₆. The dissociation constants were determined using the data points constituting the negative slopes of the Scatchard plots.

A Lipidex-1000 competition binding study was conducted by introducing unlabeled compounds into a binding medium containing 19 μM [1-¹⁴C]oleoyl-CoA. Test compounds included 8 μM free CoA and 8 μM oleoyl-CoA. A previous study indicated that micromolar concentrations of CoA were effective in stimulating microsomal DGAT activity from microspore-derived cell suspension cultures of oilseed rape 27, so it seemed logical to test the possible effect of these compounds on the binding of thioester. The presence of either free CoA or oleoyl-CoA was effective in displacing a similar quantity of radiolabeled ligand from BnDGAT1_(1–116)His₆ (Figure 3A).

Additional evidence for the interaction of BnDGAT1_(1–116)His₆ with acyl-CoA was obtained using non-denaturing gel electrophoresis. When the fragment was preincubated with an equimolar concentration of radiolabeled erucoyl-CoA followed by non-denaturing gel electrophoresis and phosphorimaging, radiolabel was extended to the position of BnDGAT1_(1–116)His₆ in the gel (Figure 3B). The rather broad band seen in the protein stain (lane 3) suggested the presence of at least two closely associated polypeptides, which probably represent different association states of BnDGAT1_(1–116)His₆ (see next section).

Analysis of the molecular mass of BnDGAT1_(1–116)His₆ under non-denaturing conditions was conducted using gel filtration chromatography of clarified lysate of ruptured bacteria expressing the N-terminal fragment (Figure 4A). Analysis of protein in the fractions eluting from the column by SDS-PAGE indicated that BnDGAT1_(1–116)His₆ eluted between the elution positions of carbonic anhydrase (29 kDa) and BSA (66 kDa).

Chemical cross-linking of BnDGAT1_(1–116)His₆ was conducted using dimethylsuberimidate (DMS) to further probe the self-associating properties of BnDGAT1_(1–116)His₆. Species generated by chemical cross-linking were resolved by SDS-PAGE and visualized by Western blotting using polyclonal antibodies which recognized an epitope in residues 21–35 in the N-terminal region of the polypeptide (Figure 4B). The control lane (lane 1) of the Western blot exhibited immunoreactive proteins with apparent molecular masses of 44 and 23 kDa, respectively. The putative 44 kDa dimer could not be visualized by Coomassie Blue staining suggesting that the immunoreactivity of the dimer was considerably greater than that of the monomer. Treatment of BnDGAT1_(1–116)His₆ with chemical cross-linking agent, however, resulted in the clear visualization of an additional band with molecular mass of 72 kDa (lane 2), which may represent tetramer, and an enhancement in the amount of putative 44 kDa dimer. The tetramer was also not visible in Coomassie Blue-stained gels. A very faint immunoreactive band of 72 kDa was also present at this position in lane 1.

Four different computer programs (HMMTOP, TMHMM, TOPPRED and TMPRED) for assessing the topology of membrane-bound proteins indicated that the hydrophilic N-terminal segment of BnDGAT1 resided on the cytosolic face of the ER. An immunochemical approach was used to provide experimental evidence for the location of the hydrophilic N-terminal segment of BnDGAT1 in microsomal vesicles. Polyclonal antibodies raised against a chemically synthesized amino acid sequence representing the peptide segment 21-LDRLHRRKSSSDSSN-35 within BnDGAT1 recognized a 56 kDa polypeptide in SDS-solubilized microsomes (Figure 5, lane 1). This molecular mass was in close agreement with the molecular mass of 56.9 kDa calculated from the deduced amino acid sequence of BnDGAT1 14. Pre-treatment of microsomes with Proteinase K, however, resulted in the loss of signal for BnDGAT1 on the Western blot (Figure 5, lane 2) suggesting that the N-terminal segment of the enzyme was digested by the proteinase in microsomal vesicles. This experiment confirmed the cytosolic location of the N-terminal segment of BnDGAT1.

Restriction enzymes were purchased from Invitrogen Canada Inc. (Mississauga, ON, Canada). PCR buffer and Taq polymerase were from Life Technologies (Burlington, ON, Canada). DNA sequencing was conducted at the University Core DNA Services of the University of Calgary (Calgary, AB, Canada). The vector pET26b(+) was from Novagen (Madison, WI, USA) and BL21 (DE3) E. coli (Stratagene, La Jolla, CA, USA). Other molecular reagents were of the highest quality and obtained from Fisher Scientific (Nepean, ON, Canada). Ni²⁺-nitrilotriacetic acid (NTA)-agarose was from Qiagen (Mississauga, ON, Canada). MALDI-TOF mass spectrometry for analysis of polypeptide molecular mass was conducted at the Institute for Biomolecular Design at the University of Alberta (Edmonton, AB, Canada). Radiolabeled oleic acid (18:1cis^Δ9; 51 mCi/mmol) and erucic acid (22:1cis^Δ13; 57 mCi/mmol) were from Amersham Canada, Oakville, ON, Canada. Acyl-CoAs were synthesized from radiolabeled fatty acids using acyl-CoA synthetase 38. Lipidex-1000 was from the Packard Instrument Company (Meriden, CT, U.S.A.). Ecolite™ (+) biodegradable scintillant was from ICN Biomedicals (Irvine, CA, U.S.A.). The Superdex 75 HR 10/30 column was from GE Healthcare, (Baie D'Urfe, QC, Canada). Proteinase K was obtained from Invitrogen Canada Inc. All other biochemicals were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada).

Most molecular genetic procedures used were adapted from Ausubel et al. 39. Analysis of the deduced amino acid sequence of BnDGAT1 indicated that residues 1–116 formed a relatively hydrophilic N-terminus 1314. The plasmid pGEM containing BnDGAT1 cDNA (GenBank accession no. AF164434) was amplified, via PCR, in the region encoding amino acid residues 1–116 using the forward and reverse primers 5'-TCATATGGCGATTTTGGATTCTGGAG-3' and 5'-GCGGCCGCATGGCTTTGTTTGAAGAT-3', respectively. The blunt ended PCR product was purified by agarose gel electrophoresis 40 and ligated into a pUC vector that had been linearized with SmaI. The ligated product was used to transform competent DH5α Escherichia coli, which were subsequently grown on β-D-thiogalactosidel/LB_amp plates. Putative transformed colonies were transferred to patch plates and after further growth, the resultant colonies were tested for the presence of the insert via restriction analysis with NdeI and NotI. A colony that tested positive for the insert via restriction analysis was sequenced to confirm the construct. The colony was grown overnight in LB_amp broth, and plasmid DNA was isolated and digested with NdeI and NotI. The products of the restriction reaction were run on an agarose gel and the band corresponding to the insert was purified from the agarose gel. The expression vector pET26b(+) was also digested and cleaned in the same manner. The insert and treated vector were ligated to each other and used to transform competent BL21 (DE3) E. coli. Selection was performed using LB_kan plates. A patch plate was generated and colony PCR was performed to test for the presence of the insert. A positive colony was isolated and propagated overnight in LB_kan broth, and the plasmid DNA was isolated and sequenced.

BL21 (DE3) cells capable of synthesizing BnDGAT1_(1–116)His₆ were grown as streak plates for 16 h at 37°C followed by inoculation into 5 mL of LB_kan. The starter liquid culture was allowed to grow for 3 h at 37°C. Five hundred milliliters of LB_kan were inoculated with the entire starter culture and the resulting mixture was incubated with vigorous shaking for 5 h at 37°C. The culture was induced with 2 mM (final concentration) IPTG and then incubated overnight at room temperature with shaking. The cells were harvested and centrifuged at 22,100 g for 10 min. After this step, all subsequent operations were conducted at 0–4°C. The supernatant was discarded, and the sediment of cells was resuspended in 10 mL of 10 mM HEPES-NaOH buffer (pH 8.0) containing 1 mM 2-mercaptoethanol and 100 mM NaCl. The suspension was passed through a French pressure cell three times at 20,000 p.s.i.. The suspension of ruptured bacteria was centrifuged at 107,000 g for 1 h to obtain the soluble subcellular fraction. The supernatant was combined with 1 mL of sedimented Ni²⁺-NTA-agarose, which was pre-equilibrated with 10 mM HEPES-NaOH (pH 8.0). The resulting suspension was incubated with constant shaking, for 30 min. The suspension was then applied to a 0.5 cm diameter polyethylene column with a porous disc at the bottom. After the immobilized nickel ion affinity gel sedimented, the column was washed with equilibration buffer and then eluted sequentially with increasing concentrations of imidazole in equilibration buffer. The column was stripped with 400 mM imidazole in equilibration buffer. Protein concentrations were determined via the Bradford 41 method using BioRad's microassay with BSA as a standard. Eluates were analyzed by SDS-PAGE 42. The separating gel was prepared using 15% monomer and 1.1% cross-linker concentration, and electrophoresis was conducted for 1.5 h at 100 V. The gel was stained with Coomassie Brilliant Blue R-250. Purified BnDGAT1_(1–116)His₆, eluted with 40 mM imidazole, was dialyzed overnight 4°C in 10 mM potassium phosphate buffer, pH 7.4 using #1 Spectra/Por Molecular porous membrane with 6,000–8,000 mol. wt. cut-off (Rancho Dominguez, CA, USA). The dialyzed BnDGAT1_(1–116)His₆ solution (~1 mg/mL) was frozen with liquid nitrogen in small aliquots in microcentrifuge tubes and stored at -80°C.

DLS was conducted using a Protein Solutions Light Scattering Instrument (DynaPro International Ltd., Crownhill, Milton Keynes, England, UK). The enzyme fragment was concentrated to about 5 mg/mL using a centrifuge-driven ultrafiltration cell equipped with 3000 mol. wt. cut-off membrane (Filtron Technology Corporation, Clinton, MA, USA). The concentrated protein solution was dialyzed overnight against 10 mM HEPES-NaOH, pH 7.4. Immediately prior to DLS, 60 μL of the protein solution was centrifuged at 12,000 g for 5 min followed by filtration through a 0.1 μm membrane. DLS was conducted at 20°C using a 12 μL cell.

Purified BnDGAT1_(1–116)His₆ was concentrated to 12 mg/mL by ultrafiltration and dialyzed overnight into various buffered environments. Initial screening for crystallization conditions was conducted using vapor diffusion and hanging drops.

Binding of radiolabeled acyl-CoA to BnDGAT1_(1–116)His₆ was conducted using a Lipidex-1000 binding assay 43. Binding assays were conducted in duplicate in 10 mM potassium phosphate buffer, pH 7.4, at 30°C with [1-¹⁴C]acyl-CoAs (oleoyl- and erucoyl-CoA) and 0.2 μM protein. Incubations were set up without protein to correct for acyl-CoA that was not adsorbed to the Lipidex-1000.

Binding of radiolabeled erucoyl-CoA to BnDGAT1_(1–116)His₆ was also analyzed using gel electrophoresis in combination with phosphorimaging based on an approach described by Engeseth et al. 44. Five microliters of BnDGAT1_(1–116)His₆ (7.5 μg) were combined with 3.3 μL of 180 μM [1-¹⁴C]erucoyl-CoA (57 mCi/mMol) in a final volume of 15 μL in a buffer environment of 10 mM potassium phosphate (pH 7.4). This incubation was set up in duplicate and included a control incubation prepared without protein. The mixture was incubated for 30 min at room temperature and then subjected to electrophoresis on a 12% separating gel (1.1% cross-linker) using Laemmli buffers 42 without SDS and without a spacer gel. Prior to sample application, 10 μL of a modified loading buffer were added to the 15 μL incubation mixture and the entire sample was subjected to gel electrophoresis. The modified loading buffer consisted of 150 μL glycerol, 40 μL of 0.5% bromophenol blue and 660 μL of 10 mM potassium phosphate buffer. Samples were not boiled prior to application to the gel. Electrophoresis was conducted at 100 V (constant voltage) until the dye front reached the bottom of the gel. One gel section containing lanes for the control and one interaction test was subjected to phosphorimaging using a Cyclone Storage Phosphor System (PerkinElmer Life and Analytical Sciences, Woodbridge, ON, Canada). The remaining gel segment was stained with Coomassie Blue R-250. The resulting phosphorimage was aligned with the gel stained for protein.

Superdex 75 HR 10/30 gel filtration chromatography was used to evaluate the native molecular mass of BnDGAT1_(1–116)His₆ in an extract prepared from E. coli overexpressing the N-terminal fragment. In this case, the E. coli producing BnDGAT1_(1–116)His₆ were resuspended in 30 mL of 100 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl. The suspension was passed three times through a French pressure cell at 20,000 p.s.i.. The suspension of ruptured bacteria was centrifuged at 13,000 g for 15 min to obtain a clarified lysate. The column was eluted at room temperature with extraction buffer at a flow rate of 0.5 ml/min using a Fast Protein Liquid Chromatography (FPLC) system (GE Healthcare Canada Inc.). One hundred microliters of extract were applied to the column. One milliliter fractions were collected and 10 μL aliquots were analyzed by SDS-PAGE. The column and SDS gel were calibrated with molecular mass markers.

BnDGAT1_(1–116)His₆ was dialyzed into 0.2 M triethanolamine-HCl, pH 8.5. Cross-linking 45 was conducted at a protein concentration of 500 μg/mL using 4 mg DMS (dihydrochloride)/mL in 0.2 M triethanolamine-HCl buffer (pH 8.5) for 3 h at room temperature. DMS was dissolved in buffer immediately before use. Cross-linking reactions were quenched with 2 × SDS loading buffer and boiled for 5 min prior to SDS-PAGE. Western blotting was conducted according to Nykiforuk et al. 13 using polyclonal antibodies raised against a peptide of 15 of amino acid residues (21-LDRLHRRKSSSDSSN-35) representing a segment within the N-terminal region of BnDGAT1.

Four programs from the Expasy Molecular Biology Server 46 were used for topology predictions of the deduced BnDGAT1 amino acid sequence: HMMTOP 47, TMHMM 48, TOPPRED 49 and TMPRED 50.

Microspore-derived cell suspension cultures of B. napus L. cv Jet Neuf were maintained according to Orr et al. 51. Microsomes sedimenting at 10,000–100,000 g were prepared from 1 g of frozen cultured cells according to Byers et al. 27. Microsomes were resuspended in 10 mM HEPES-NaOH buffer (pH 7.4) and homogenized with a Potter Elvehjem homogenizer, and then centrifuged for 20 min at 220,000 g at 4°C. The pellet was resuspended in 1 mL HEPES-NaOH buffer (pH 7.4) and the protein content was determined.

Microsomes containing 1 mg protein were treated with 0.01 mg Proteinase K for 30 min on ice according to Morimoto et al. 52. The reaction was terminated by adding 0.1 mg phenylmethylsulfonyl fluoride per mg Proteinase K followed by 5 min of additional incubation on ice. Samples were diluted to 1 ml with 10 mM HEPES-NaOH buffer (pH 7.4) and centrifuged at 220,000 g for 20 min at 4°C. The resulting pellets were resuspended in 150 μl SDS loading buffer, boiled for 5 min and 25 μL were subjected to SDS-PAGE (12% total monomer and 1.1% cross-linker concentration). Western blotting was performed according to Nykiforuk et al. 13.