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.

RNA helicases represent a large family of proteins that have been detected in almost all biological systems where RNA plays a central role. They are ubiquitously distributed over a wide range of organisms and are involved in nuclear and mitochondrial splicing processes, RNA editing, rRNA processing, translation initiation, nuclear mRNA export, and mRNA degradation. RNA helicases are described as essential factors in cell development and differentiation, and some of them play a role in transcription and replication of viral single-stranded RNA genomes. Comparisons of the conserved sequences reveal a close relationship between them and suggest that these proteins might be derived from a common ancestor. Biochemical studies have revealed a strong dependence of the unwinding activity on ATP hydrolysis. Although RNA helicase activity has only been demonstrated for a few examples yet, it is generally believed that all members of the largest subgroups, the DEAD and DEAH box proteins, exhibit this activity.

The strong overexpression or complete deletion of a gene gives only limited information about its control over a certain phenotype or pathway. Gene function studies based on these methods are therefore incomplete. To effect facile manipulation of gene expression across a full continuum of possible expression levels, we recently created a library of mutant promoters. Here, we provide the detailed characterization of our yeast promoter collection comprising 11 mutants of the strong constitutive Saccharomyces cerevisiae TEF1 promoter. The activities of the mutant promoters range between about 8% and 120% of the activity of the unmutated TEF1 promoter. The differences in reporter gene expression in the 11 mutants were independent of the carbon source used, and real-time PCR confirmed that these differences were due to varying levels of transcription (i.e., caused by varying promoter strengths). In addition to a CEN/ARS plasmid-based promoter collection, we also created promoter replacement cassettes. They enable genomic integration of our mutant promoter collection upstream of any given yeast gene, allowing detailed genotype-phenotype characterizations. To illustrate the utility of the method, the GPD1 promoter of S. cerevisiae was replaced by five TEF1 promoter mutants of different strengths, which allowed analysis of the impact of glycerol 3-phosphate dehydrogenase activity on the glycerol yield.In both functional genomics and metabolic engineering, strategies for fine-tuning gene expression are required to study the control exerted by target genes on phenotypes or metabolic fluxes of interest. Traditionally, gene expression varies among three conditions: (i) the wild type, (ii) the gene knockout, and (iii) strong overexpression of the target gene. However, several examples where the optimization of target phenotypes was achieved by moderate rather than strong multicopy expression of a target gene have been given (12,13,30). These examples highlight the need for tools that enable the fine-tuning and precise control of gene expression in Saccharomyces cerevisiae.Several strategies have been explored to obtain a graded target gene expression. One strategy is to clone native promoters of various strengths to drive the expression of a desired gene (34). However, this method is not robust, since endogenous promoters may be subject to different regulation modalities, even despite their designation as "constitutive." Moreover, a native promoter of a given desired strength may be unavailable or difficult to identify. A second strategy uses vectors of different copy numbers to adjust gene expression levels (12,13,30). This approach is limited by the availability of plasmids of any given desired copy number. Other problems with this technique are the metabolic burden associated with maintenance of high-copy number plasmids and the cell-cell heterogeneity in expression caused by copy number variance between single cells of a culture. A third strategy for modulating gene expression has been to titrate an inducible promoter sy...

The enzyme 3-hydroxy-3-methylglutaryl-co-enzyme-A (HMG-CoA) reductase is known as the rate limiting enzyme in early sterol biosynthesis in eukaryotic cells. To eliminate this regulation in the yeast Saccharomyces cerevisiae, a truncated HMG1 gene, producing a form of the enzyme that lacks the membrane-binding region (i.e. amino acids 1-552), was constructed and overexpressed in this yeast. The transformed strains accumulated large amounts of the sterol precursor squalene, while the levels of ergosterol and a number of other sterol compounds were only slightly elevated. These findings suggest that HMG-CoA reductase is not the only rate-limiting step in sterol synthesis and its overexpression cannot significantly influence this pathway beyond the sterol precursor squalene.