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Edited by Christopher R. Somerville, Carnegie Institution of Washington, Stanford, CA, and approved March 31, 2000 (received for review February 15, 2000)
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- Figure 1
Metabolism of 14C-labeled PC into the neutral lipid fraction by plant microsomes. (A) Microsomes from developing seeds of sunflower, R. communis, and C. palaestina were incubated for 80 min at 30°C with PC (8 nmol) having oleic acid in its sn-1 position, and 14C-labeled oleic, ricinoleic, or vernolic acid in its sn-2 position. Radioactivity incorporated into TAG (open bars), DAG (filled bars), and unesterified fatty acids (hatched bars) is shown as percentage of added labeled substrate. (B) Synthesis in vitro of TAG carrying two vernoloyl and one [14C]ricinoleoyl group by microsomes from R. communis. The substrates added were unlabeled divernoloyl-DAG (5 nmol), together with either sn-1-oleoyl-sn-2-[14C]ricinoleoyl-DAG (0.4 nmol, 7,700 dpm/nmol) or sn-1-oleoyl-sn-2-[14C]ricinoleoyl-PC (0.4 nmol, 7,700 dpm/nmol). The microsomes were incubated with the substrates for 30 min at 30°C as described in Materials and Methods. The data shown are the average of two experiments.
- Figure 2
PDAT activity in yeast microsomes, as visualized by autoradiogram of neutral lipid products separated on TLC. Microsomal membranes from the wild-type yeast strain FY1679 (lanes 1–3), a congenic yeast strain [FVKT004–04C(AL)] that is disrupted for YNR008w (lane 4), or the same disruption strain transformed with the plasmid pUS1, containing the YNR008w gene behind its native promoter (lane 5), were assayed for PDAT activity. As substrates, we used 2 nmol of sn-1-oleoyl-sn-2-[14C]ricinoleoyl-PC together with either 5 nmol of dioleoyl-DAG (lanes 2, 4, and 5) or rac-oleoyl-vernoloyl-DAG (lane 3). The enzymatic assay and lipid analysis were performed as described in Materials and Methods. Abbreviations: 1-OH-TAG, monoricinoleoyl-TAG; 1-OH-1-ep-TAG, monoricinoleoyl-monovernoloyl-TAG; OH-FA, unesterified ricinoleic acid.
- Figure 3
Lipid content (A and B) and PDAT activity (C) in PDAT-overexpressing yeast cells. The PDAT gene in the plasmid pUS4 was overexpressed from the galactose-induced GAL1 promoter in the wild-type strain W303-1A (10). Its expression was induced after 2 h (A) or 25 h (B) of growth as described in Materials and Methods. The amount of TAG (open bar), polar lipids (hatched bar), sterol esters (filled bar), and other lipids (striped bar) of these cells is presented as μmol of fatty acids per mg of dry weight. The data shown are the mean values of results with three independent yeast cultures. (C) In vitro synthesis of TAG by microsomes prepared from yeast cells, cultivated as in A, containing either the empty vector (vector) or the PDAT plasmid (+PDAT). The substrate lipids dioleoyl-DAG (2.5 nmol) and sn-1-oleoyl-sn-2-[14C]oleoyl-PC (2 nmol) were added to aliquots of microsomes, which were then incubated for 10 min at 30°C. The results shown are the mean values of two experiments.
- Figure 4
Substrate specificity of yeast PDAT. (A) sn-position specificity of yeast PDAT regarding the acyl donor substrate. Dioleoyl-DAG (2.5 nmol) together with 4 nmol of sn-1-[14C]oleoyl-sn-2-[14C]oleoyl-PC (di-[14C]-PC), sn-1-[14C]oleoyl-sn-2-oleoyl-PC (sn1-[14C]-PC), or sn-1-oleoyl-sn-2-[14C]oleoyl-PC (sn2-[14C]-PC) was used as substrate. (B) Specificity of yeast PDAT regarding the phospholipid headgroup and the acyl composition of the phospholipid as well as the DAG. Dioleoyl-DAG (2.5 nmol) together with 4 nmol of sn-1-oleoyl-sn-2-[14C]oleoyl-PC (oleoyl-PC), sn-1-oleoyl-sn-2-[14C]oleoyl-PE (oleoyl-PE), sn-1-oleoyl-sn-2-[14C]ricinoleoyl-PC (ricinoleoyl-PC), or sn-1-oleoyl-sn-2-[14C]vernoloyl-PC (vernoloyl-PC) was used as substrate. In the experiments presented in the two bars to the far right, 2.5 nmol of monoricinoleoyl-DAG (ricinoleoyl-DAG) or monovernoloyl-DAG (vernoloyl-DAG) were used together with 4 nmol of sn-1-oleoyl-sn-2-[14C]oleoyl-PC. Microsomes from W303-1A cells overexpressing the PDAT gene, as described in Fig. 3A, were incubated at 30°C for 10 min (A) or 90 min (B). The synthesis of radiolabeled TAG (solid bars) and lyso-PC (LPC, open bars) are the mean values of two experiments.
- Figure 5
Evolutionary dendrogram showing LCAT- and PDAT-related proteins from different eukaryotes. The dendrogram was calculated from aligned protein sequences corresponding to amino acid residues 174–335 in yeast PDAT. The clustalx multiple alignment program (28) was used with default settings to align the sequences and compute pairwise alignment scores. An unrooted tree was then obtained from these scores by using the neighbor-joining method (29), with correction for multiple substitutions and exclusion of gapped positions.
Phospholipid:diacylglycerol acyltransferase: An enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants
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