Understanding the roles of diacylglycerol acyltransferases in triacylglycerol biosynthesis and storage

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Triacylglycerol (TG) is quantitatively the most important storage form of metabolic energy for most living organisms. TGs consist of a glycerol backbone to which three fatty acids are attached. Because they are highly reduced and anhydrous, TGs are capable of providing much more energy than carbohydrate or protein. Eukaryotic organisms store TGs in cytosolic lipid droplets that can be metabolized during periods of nutrient deprivation when energy requirements are not being satisfied. Plants store large quantities of TGs in seeds that are used as energy for seedling growth and development. TG is synthesized by an enzyme called diacylglycerol acyltransferase (DGAT) which uses diacylglycerol and fatty acyl CoA as substrates. Two DGAT enzymes that share no sequence similarity have now been identified: DGAT1 and DGAT2. Although these two enzymes catalyze the same biochemical reaction in vitro, they appear to not have redundant functions in cells. DGAT1 and DGAT2 are proposed to produce distinct pools of TG. Both enzymes are present in the endoplasmic reticulum in cells; however, DGAT2 is unique in that it also translocates to lipid droplets, the organelle in which TG is stored. The presence of DGAT2 on lipid droplets is thought to allow for localized TG synthesis, leading to lipid droplet growth. Little is known about how the DGAT enzymes function in cells to promote the synthesis and storage of TG. Therefore, an integrated approach using biochemical and molecular techniques in parallel with proteomics and lipidomics will be used to understand how DGAT1 and DGAT2 work to synthesize TG. The specific objectives of the proposed research program are to: (1) Identify the lipid droplet targeting/binding domain of DGAT2, (2) Characterize the fatty acid signals involved in protein translocation to lipid droplets, (3) Determine if DGAT1 and DGAT2 produce distinct pools of TGs, and (4) Compare and contrast the enzymatic properties of DGAT1 and DGAT2 using a yeast expression system. The proposed studies will address fundamental questions in lipid metabolism and provide new insights into the molecular mechanism responsible for TG synthesis that may be of interest to both the pharmaceutical and agricultural industries. Due to the alarming increase in the prevalence of obesity in Canada, there is intense interest of pharmaceutical companies in Canada to develop therapeutic compounds to reduce TG synthesis and storage. In plants, TG is found in large quantities in oil seeds such as canola which represents a valuable resource for dietary consumption and industrial uses in Canada. Since many of the pathways for TG synthesis are conserved from mammals to plants, there is also the potential for knowledge transfer of our research to the agricultural sector with the goal of modifying TG production in oil seed crops. This could have a significant impact on plant biotechnology and the agricultural industry in Canada.