Triacylglycerol (TAG) is known to be synthesized in a reaction that uses acyl-CoA as acyl donor and diacylglycerol (DAG) as acceptor, and which is catalyzed by the enzyme acyl-CoA:diacylglycerol acyltransferase. We have found that some plants and yeast also have an acyl-CoA-independent mechanism for TAG synthesis, which uses phospholipids as acyl donors and DAG as acceptor. This reaction is catalyzed by an enzyme that we call phospholipid:diacylglycerol acyltransferase, or PDAT. PDAT was characterized in microsomal preparations from three different oil seeds: sunflower, castor bean, and Crepis palaestina. We found that the specificity of the enzyme for the acyl group in the phospholipid varies between these species. Thus, C. palaestina PDAT preferentially incorporates vernoloyl groups into TAG, whereas PDAT from castor bean incorporates both ricinoleoyl and vernoloyl groups. We further found that PDAT activity also is present in yeast microsomes. The substrate specificity of this PDAT depends on the head group of the acyl donor, the acyl group transferred, and the acyl chains of the acceptor DAG. The gene encoding the enzyme was identified. The encoded PDAT protein is related to lecithin:cholesterol acyltransferase, which catalyzes the acyl-CoA-independent synthesis of cholesterol esters. However, budding yeast PDAT and its relatives in fission yeast and Arabidopsis form a distinct branch within this protein superfamily, indicating that a separate PDAT enzyme arose at an early point in evolution.oil seeds ͉ phospholipids T riacylglycerol (TAG) is the most common lipid-based energy reserve in nature. The main pathway for synthesis of TAG is believed to involve three sequential acyl-transfers from acylCoA to a glycerol backbone (1, 2). For many years, acyl-CoA:diacylglycerol acyltransferase (DAGAT), which catalyzes the third acyl transfer reaction, was thought to be the only enzyme specifically involved in TAG synthesis. It acts by diverting diacylglycerol (DAG) from membrane lipid synthesis into TAG (2). Genes encoding this enzyme were recently identified both in mice (3) and in plants (4-6), and the encoded proteins were shown to be homologous to acyl-CoA:cholesterol acyltransferase (ACAT). It was also recently reported that another DAGAT exists in the oleaginous fungus Mortierella ramanniana, which is unrelated to the mouse DAGAT, the ACAT gene family, or to any other known gene (K. Lardizabal, personal communication). In addition to these acyl-CoA-dependent enzymes, recent work has shown that, in microsomal preparations from oil seeds, TAG synthesis can also occur in the absence of acyl-CoA (7, 8). However, the enzyme involved in this acyl-CoA-independent synthesis of TAG has not yet been identified in any organism.In this paper, we characterize the acyl-CoA-independent synthesis of TAG in plants, and we conclude that it is mediated by an enzyme that we call phospholipid:diacylglycerol acyltransferase (PDAT). This enzyme is proposed to be involved in the accumulation of high levels of hydroxylated fatty acid (...

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.Lipids are important storage compounds in plants, animals, and fungi. The main storage lipids in eukaryotes are triacylglycerol (TAG) 1 and steryl esters (1). Storage lipids are usually found within special organelles known as lipid particles or lipid bodies (2). In yeast, these lipid bodies accumulate during stationary phase, and they can constitute up to 70% of the total lipid content of the cell (3-5). Several lipid-metabolizing enzymes are preferentially localized to the lipid bodies in yeast, and it has therefore been proposed that they do not solely serve as a depot for lipids but instead may have a more complex role in lipid biosynthesis, metabolism, degradation, and trafficking (6).Steryl esters, the esterified form of sterols linked to a long chain fatty acid, are synthesized by the enzyme acyl-CoA:sterol acyltransferase (ASAT) which is encoded by the duplicated ARE1 and ARE2 genes in the yeast Saccharomyces cerevisiae (7-9). Related genes encoding enzymes with ASAT activity exist also in higher eukaryotes (10, 11). It has been shown that the other major storage lipid, TAG, can be synthesized in at least two different ways. One is an acyl-CoA-dependent reaction that is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT). Two different DGATs are known to exist in eukaryotes. One DGAT, also known as DGAT1, was first described in mammals but is also found in plants and is related to ASAT (12,13). In addition, a second DGAT, DGAT2, was recently identified in the fungus Mortiella ramanniana (14). DGAT2 is not related to any previously known enzyme, but genes encoding homologues of DGAT2 exist in other eukaryotes (14,15). One such gene is the DGA1 gene in yeast (open reading frame YOR245c). In addition to the reaction catalyzed by the two types of DGATs, an acyl-CoA-independent pathway for TAG synthesis was also recently discovered in plants and yeast (16). This pathway involves the novel enzyme phospholipid: diacylglycerol acyltransferase (PDAT), which can synthesize TAG from phospholipids and diacylglycerol (16). PDAT is distantly related to the mammalian enzyme lecithin:cholesterol acyltr...

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.

To investigate cation adaptation and homoeostasis in Aspergillus nidulans, two transcription-factor-encoding genes have been characterized. The A. nidulans orthologue crzA of the Saccharomyces cerevisiae CRZ1 gene, encoding a transcription factor mediating gene regulation by Ca(2+), has been identified and deleted. The crzA deletion phenotype includes extreme sensitivity to alkaline pH, Ca(2+) toxicity and aberrant morphology connected with alterations of cell-wall-related phenotypes such as reduced expression of a chitin synthase gene, chsB. A fully functional C-terminally GFP (green fluorescent protein)-tagged form of the CrzA protein is apparently excluded from nuclei in the absence of added Ca(2+), but rapidly accumulates in nuclei upon exposure to Ca(2+). In addition, the previously identified sltA gene, which has no identifiable homologues in yeasts, was deleted, and the resulting phenotype includes considerably enhanced toxicity by a number of cations other than Ca(2+) and also by alkaline pH. Reduced expression of a homologue of the S. cerevisiae P-type ATPase Na(+) pump gene ENA1 might partly explain the cation sensitivity of sltA-null strains. Up-regulation of the homologue of the S. cerevisiae vacuolar Ca(2+)/H(+) exchanger gene VCX1 might explain the lack of Ca(2+) toxicity to null-sltA mutants, whereas down-regulation of this gene might be responsible for Ca(2+) toxicity to crzA-null mutants. Both crzA and sltA encode DNA-binding proteins, and the latter exerts both positive and negative gene regulation.

Programmed cell death (PCD) is executed by proteases, which cleave diverse proteins thus modulating their biochemical and cellular functions. Proteases of the caspase family and hundreds of caspase substrates constitute a major part of the PCD degradome in animals. Plants lack close homologues of caspases, but instead possess an ancestral family of cysteine proteases, metacaspases. Although metacaspases are essential for PCD, their natural substrates remain unknown. Here we show that metacaspase mcII-Pa cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), during both developmental and stress-induced PCD. TSN knockdown leads to activation of ectopic cell death during reproduction, impairing plant fertility. Surprisingly, human TSN (also known as p100 or SND1), a multifunctional regulator of gene expression, is cleaved by caspase-3 during apoptosis. This cleavage impairs the ability of TSN to activate mRNA splicing, inhibits its ribonuclease activity and is important for the execution of apoptosis. Our results establish TSN as the first biological substrate of metacaspase and demonstrate that despite the divergence of plants and animals from a common ancestor about one billion years ago and their use of distinct PCD pathways, both have retained a common mechanism to compromise cell viability through the cleavage of the same substrate, TSN.