Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Comparison of HPLC/PDA profiles of yeast and tung oil triglycerides. (A) Wild-type yeast cells harboring an empty expression plasmid (WT+pYes3) were cultivated in the presence of tung oil and a nonspecific lipase, then cells were harvested, washed extensively, and cellular lipids extracted and analyzed by high performance liquid chromatography and photodiode array detection (HPLC/PDA) as described in the legend of Figure 4. Triacylglycerols (TAGs) were separated by HPLC and the PDA detector monitored UV absorbance from 210 to 345 nm. Peaks are labeled according to the fatty acid composition of each TAG molecule, although positional information of each fatty acyl side chain was not determined. Inset shows an overlay of the extracted 271 nm traces obtained from PDA analysis of yeast lipids (A) or a tung oil standard (B). Panels are representative of three independent experiments. (B) HPLC/PDA analysis of a tung oil standard. Inset shows the UV absorbance pattern of eleostearic acid, showing the characteristic spectrum with 3 peaks and a a_max of 271 nm.
• Supplemental Figure 2 - Identification of TAGs by LC/MS. The fatty acid composition of each TAG species was determined by liquid chromatography/mass spectrometry (LC/MS) analysis of fragmentation peaks corresponding to the loss of the sn-1 or sn-3 fatty acid (DAG fragments) and the loss of both the 1- and 3-fatty acids (MAG fragment). As an example, the mass spectrum associated with the peak labeled EOPo in the HPLC/PDA chromatograms (Figure 4B and Supplemental Figure 3B and D) is shown. This peak had an MH⁺ ion of m/z 853, which corresponded to three potential TAGs: ELP, EOPo, or LLPo, excluding positional isomers. The DAG fragment ions associated with the peak had masses of m/z 599.7 and 571.7, which correspond to EO (with a loss of Po) and EPo (with a loss of O), respectively. Based on these data, a positional isomer of EOPo is the only possible structure. While this method was useful for identifying the fatty acyl composition of the TAG species, it was not possible to determine the positional specificity of fatty acids on the glycerol backbone because TAGs containing eleostearic acid did not fragment extensively to produce the monoglyceride, s-2 labeled species.
• Supplemental Figure 3 - Comparison of native, myc-tagged, and GFP-tagged DGAT1 and DGAT2 activities in yeast cells. Mutant yeast cells (MUT) that lack the ability to synthesize appreciable amounts of TAG were transformed with plasmids containing either native, myc-tagged, or GFP-tagged DGAT1 or DGAT2. Cells were cultivated in the presence of tung oil and a nonspecific lipase, then cells were harvested and lipids extracted for identification of eleostearoyl-containing lipid compounds. Strain and plasmid combinations are shown along the bottom of (A) and (C) and to the right of (B) and (D). (A and C) Percentage of eleostearic acid in mutant yeast cells complemented with various DGAT1 (A) or DGAT2 (C) enzymes, as determined by gas chromatography (top panels of A and C), and thin-layer chromatography (bottom panels of A and C) showing the various lipid classes present in the yeast lipid extracts. Positions of lipid standards are shown to the left: phospholipids (PL), diacylglycerol (DAG), free fatty acids (FFA), triacylglycerols (TAG), and sterol esters (ST). Samples are representative of three independent experiments. (B and D) HPLC/PDA analysis of yeast lipids. Lipids were extracted from yeast cells expressing various DGAT1 (B) or DGAT2 (D) enzymes then TAGs were separated and analyzed by an HPLC equipped with a photodiode array (PDA) detector. The PDA monitored UV absorbance (210 to 345 nm) of compounds during separation, and eleostearoyl-containing TAGs were identified by the characteristic UV spectrum of eleostearic acid. Each chromatogram shows the extracted 271 nm absorbance trace (ƒtalpha;_max of eleostearic acid), and TAG peaks are labeled according to the three fatty acyl side chains. Fatty acid abbreviations are: eleostearic (E), linoleic (L), oleic (O), palmitic (P), palmitoleic (Po), stearic (S). Note the similarity of HPLC/PDA chromatograms for the native, myc-tagged and GFP-tagged DGAT1 and DGAT2 enzymes. Each chromatogram is representative of three independent experiments.
• Supplemental Figure 4 - Subcellular localization of DGAT1 and DGAT2 in tobacco BY-2 cells. BY-2 cells were transformed transiently with either myc-DGAT1 or myc-DGAT2, fixed in formaldehyde at 4 h after biolistic bombardment, and then processed for immunofluorescence CLSM. Hatched boxes represent the portion of the cell shown at higher magnification in the panels below. The yellow/orange color in the merged images indicates co-localization of expressed myc-DGAT1 or myc-DGAT2 and endogenous ER stained with DiOC₆ in the same cells; white arrowheads also indicate co-localizations. Black arrowheads indicate regions of DiOC₆-stained ER that contain relatively low immunofluorescence attributable to myc-DGAT1 or myc-DGAT2. Note that the large punctate fluorescent structures observed in micrographs of DiOC₆-stained cells (see asterisks) are likely Golgi and endosomes since DiOC₆ is known to label these and other organelles (Sabnis et al., 1997). We discounted this background DiOC₆ staining given that myc-DGAT1 and myc-DGAT2 did not localize at steady-state to these organelles (compare the DiOC₆ punctate fluorescence with the immunofluorescence patterns attributable to myc-DGAT1 or myc-DGAT2 in the cells). Bar = 10 μm.
• Supplemental Figure 5 - Subcellular localization of co-expressed myc-DGAT1 and HA-DGAT2 in tobacco BY-2 cells. BY-2 cells were co-transformed with myc-DGAT1 and HA-DGAT2, fixed in formaldehyde at 4 h after biolistic bombardment, and then processed for immunofluorescence CLSM. Hatched boxes represent the portion of the cell shown at higher magnification in the panels below. Note that the fluorescence patterns attributable to co-expressed myc-DGAT1 and HA-DGAT2 in the same cell are different; compare the non-overlapping red and green fluorescence, respectively, in the merged image. Endogenous ER in the same co-transformed cell was stained with ConA-conjugated to Alexa 594; fluorescence attributable to ConA staining is pseudocolored white. Bar = 10 μm.
• Supplemental Figure 6 - Subcellular localization of DGAT1, DGAT2 and calnexin in tobacco BY-2 cells. BY-2 cells were transformed transiently with either myc-DGAT1 or myc-DGAT2, fixed in formaldehyde at 4 h after biolistic bombardment, and then processed for immunofluorescence CLSM. Hatched boxes represent the portion of the cell shown at higher magnification in the panels below. Note that the punctate fluorescence patterns attributable to expressed myc-DGAT1 or myc-DGAT2 and endogenous calcium-binding ER membrane protein calnexin in the same cell are mostly different (compare the non-overlapping green and red fluorescence, respectively, in the merged image; arrowheads indicate non-colocalizations). The yellow/orange color in the merged images indicates other regions of the ER where expressed myc-DGAT1 or myc-DGAT2 and endogenous calnexin co-localize. Compare also the localization of either expressed myc-DGAT1 or myc-DGAT2, endogenous calnexin, and/or ConA-stained ER in the same cells (see also Figure 5). Fluorescence attributable to endogenous ER stained with ConA-conjugated to Alexa 594 is pseudocolored white. Bar = 10 μm.
• Supplemental Figure 7 - Co-localization of GFP-Cf9-DGAT2 and GFP-Cf9-DGAT2+13 with the Golgi marker protein RFP-BS14a in onion epidermal cells. Onion epidermal peels were co-bombarded with DNA encoding either GFP-Cf9-DGAT2 or GFP-Cf9-DGAT2+13 and RFP-BS14a, consisting of the red fluorescent protein (RFP) fused to the Nterminus of the Arabidopsis Golgi-localized soluble N-ethyl-maleimide sensitive factor attachment protein receptor (SNARE) BS14a (Uemura et al., 2004). Cells were then incubated for 8 hours in the dark and visualized using either fluorescence or brightfield microscopy. Each representative micrograph is labeled at the top left with either the name of the transiently-expressed GFP-Cf9 fusion protein or RFP-BS14a. The yellow/orange color in the merged images indicates where co-expressed GFP-Cf9-DGAT2 or GFP-Cf9-DGAT2+13 co-localized in Golgi with RFP-BS14a. Also shown are the corresponding brightfield micrographs of the co-transformed cells. Arrowheads indicate examples of obvious co-localizations. Bar = 100 μm.
• Supplemental Figure 8 - Functional and cellular analysis of Arabidopsis DGAT1 and DGAT2. (A) Comparison of Arabidopsis (At) and tung (Vf) DGAT1 and DGAT2 enzyme activities after expression in mutant yeast cells (MUT) that lack the ability to synthesize triacylglycerols (TAGs). Strain and plasmid combinations are shown along the bottom. Cells were cultivated in the presence of tung oil and a nonspecific lipase, then cells were harvested and lipids extracted for identification of eleostearoyl-containing compounds. Upper panel shows the percentage of eleostearic acid in yeast cells complemented with various DGAT1 or DGAT2 enzymes, as determined by gas chromatography with flame ionization detection (GC/FID). Bottom panel shows a thin layer chromatography (TLC) separation of various lipid classes. Positions of lipid standards are shown to the left: phospholipids (PL), diacylglycerol (DAG), free fatty acids (FFA), triacylglycerols (TAG), and sterol esters (ST). Note the reduction of eleostearic acid (top panel) and absence of TAGs (bottom panel) in cells expressing Arabidopsis DGAT2 (MUT+AtDGAT2). (B) HPLC/PDA analysis of lipids extracted from mutant yeast cells expressing tung DGAT1 (MUT+VfDGAT1) or Arabidopsis DGAT1 (MUT+AtDGAT1). Each chromatogram represents the 271 nm absorbance trace (a_max of eleostearic acid) extracted from the complete PDA dataset. Each TAG peak is labeled according to the three fatty acyl side chains, although stereospecific positions are not known. Fatty acid abbreviations are: eleostearic (E), linoleic (L), oleic (O), palmitic (P), palmitoleic (Po), stearic (S). (C) Subcellular localization of Arabidopsis DGAT1 and DGAT2 in tobacco BY-2 cells. BY-2 cells were transformed transiently with either myc-epitope-tagged Arabidopsis DGAT1 (myc-AtDGAT1) or DGAT2 (myc-AtDGAT2), fixed in formaldehyde at 4 h after biolistic bombardment, and then processed for immunofluorescence CLSM. Hatched boxes represent the portion of the cell shown at higher magnification in the panels below. The yellow/orange color in the merged images indicates co-localization of expressed myc-AtDGAT1 or myc-AtDGAT2 and endogenous ER stained with ConA-conjugated to Alexa 594 in the same cells; arrowheads also indicate distinct regions of the ConA-stained ER where myc-AtDGAT1 was enriched. Note that myc-AtDGAT2 is not found in distinct regions of the ER, but rather is distributed throughout the entire ER network. Bar = 10 μm.
• Supplemental Figure 9 - Alignment of DGAT1 and DGAT2 protein sequences. The protein sequences of known and putative DGAT genes were aligned using the CLUSTALX program (version 1.81, Thompson et al., 1997) using default settings. Each plant species is represented by either DGAT1 or DGAT2, or both, depending on availability of database sequences. Proteins used in the analysis included DGAT1 and/or DGAT2 sequences from tung (Vf), Arabidopsis (At), Brassica napus (Bn), cotton (Gh), rice (Os), soybean (Gm), tobacco (Nt), wheat (Ta), human (Hs), and yeast (Sc).