Saccharomyces cerevisiae phospholipid:diacylglycerol acyl transferase (PDAT) devoid of its membrane anchor region is a soluble and active enzyme retaining its substrate specificities.

A N-terminal deleted version of the Saccharomyces cerevisiae phospholipid:diacylglycerol acyltransferase (ScPDAT), lacking the predicted membrane-spanning region, was fused in frame with alpha-factor secretion signal and expressed in Pichia pastoris under the control of the methanol inducible alcohol oxidase promoter. This resulted in a truncated, soluble and highly active PDAT protein secreted into the culture medium of the recombinant cells. The soluble as well as native membrane bound enzymes was shown to be glycosylated and extensive deglycosylation severely lowered the activity. The production of a soluble and extracellular PDAT allowed us to investigate substrate preferences of the enzyme without interference of endogenous lipids and enzymes. Similar to the membrane bound counterpart, the highest activity was achieved with acyl groups at sn-2 position of phosphatidylethanolamine as acyl donor and 1,2-diacylglycerols as acyl acceptor. The soluble enzyme was also able to catalyze, at a low rate, a number of transacylation reactions between various neutral lipids and between polar lipids and neutral lipids others than diacylglycerols, including acylation of long chain alcohols.

Lipids in grain tissues of oat (Avena sativa): differences in content, time of deposition, and fatty acid composition.

Oat (Avena sativa) is unusual in comparison with other cereals since there are varieties with up to 18% oil content. The lipid content and fatty acid composition in different parts of the grain during seed development were characterized in cultivars Freja (6% oil) and Matilda (10% oil), using thin-layer and gas chromatography, and light and electron microscopy. The majority of lipids (86-90%) were found in the endosperm. Ninety-five per cent of the higher oil content of cv. Matilda compared with cv. Freja was due to increased oil content of the endosperm. Up to 84% of the lipids were deposited during the first half of seed development, when seeds where still green with a milky endosperm. Microscopy studies revealed that whereas oil bodies of the embryo and scutellum still contained a discrete shape upon grain maturation, oil bodies of the endosperms fused upon maturation and formed smears of oil.

Cloning and functional characterization of a phospholipid:diacylglycerol acyltransferase from Arabidopsis.

A new pathway for triacylglycerol biosynthesis involving a phospholipid:diacylglycerol acyltransferase (PDAT) was recently described (Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S, [2000] Proc Natl Acad Sci USA 97: 6487-6492). The LRO1 gene that encodes the PDAT was identified in yeast (Saccharomyces cerevisiae) and shown to have homology with animal lecithin:cholesterol acyltransferase. A search of the Arabidopsis genome database identified the protein encoded by the At5g13640 gene as the closest homolog to the yeast PDAT (28% amino acid identity). The cDNA of At5g13640 (AtPDAT gene) was overexpressed in Arabidopsis behind the cauliflower mosaic virus promoter. Microsomal preparations of roots and leaves from overexpressers had PDAT activities that correlated with expression levels of the gene, thus demonstrating that this gene encoded PDAT (AtPDAT). The AtPDAT utilized different phospholipids as acyl donor and accepted acyl groups ranging from C10 to C22. The rate of activity was highly dependent on acyl composition with highest activities for acyl groups containing several double bonds, epoxy, or hydroxy groups. The enzyme utilized both sn-positions of phosphatidylcholine but had a 3-fold preference for the sn-2 position. The fatty acid and lipid composition as well as the amounts of lipids per fresh weight in Arabidopsis plants overexpressing AtPDAT were not significantly different from the wild type. Microsomal preparations of roots from a T-DNA insertion mutant in the AtPDAT gene had barely detectable capacity to transfer acyl groups from phospholipids to added diacylglycerols. However, these microsomes were still able to carry out triacylglycerol synthesis by a diacylglycerol:diacylglycerol acyltransferase reaction at the same rate as microsomal preparations from wild type.

Storage lipid synthesis is non-essential in yeast.

Steryl esters and triacylglycerol (TAG) are the main storage lipids in eukaryotic cells. In the yeast Saccharomyces cerevisiae, these storage lipids accumulate during stationary growth phase within organelles known as lipid bodies. We have used single and multiple gene disruptions to study storage lipid synthesis in yeast. Four genes, ARE1, ARE2, DGA1, and LRO1, were found to contribute to TAG synthesis. The most significant contribution is made by DGA1, which encodes a novel acyl-CoA:diacylglycerol acyltransferase. Two of the genes, ARE1 and ARE2, are also involved in steryl ester synthesis. A yeast strain that lacks all four genes is viable and has no apparent growth defects under standard conditions. The strain is devoid of both TAG and steryl esters, and fluorescence microscopy revealed that it also lacks lipid bodies. We conclude that neither storage lipids nor lipid bodies are essential for growth in yeast.