Triacylglycerol lipases of the yeast

All eukaryotes including the yeast contain a lipid storage compartment which is named lipid particle, lipid droplet or oil body. Lipids accumulating in this subcellular fraction serve as a depot of energy and building blocks for membrane lipid synthesis. In the yeast, the major storage lipids are triacylglycerols (TGs) and steryl esters (SEs). An important step in the life cycle of these non-polar lipids is their mobilization from their site of storage and channeling of their degradation components to the appropriate metabolic pathways. A key step in this mobilization process is hydrolysis of TG and SE which is accomplished by lipases and hydrolases. In this review, we describe our recent knowledge of TG lipases from the yeast based on biochemical, molecular biological and cell biological information. We report about recent findings addressing the versatile role of TG lipases in lipid metabolism, and discuss non-polar lipid homeostasis and its newly discovered links to various cell biological processes in the yeast.     

Triacylglycerol biosynthesis in bovine liver and subcutaneous adipose tissue.

1. Adipose tissue from Angus and Brahman steers incubated with [1-14C]palmitate in the absence and presence of glucose exhibited a greater rate of lipid production than liver (P < 0.05). 2. Homogenates of adipose tissue used in the glycerol-3-phosphate acyltransferase assay exhibited a greater glycerolipid specific activity (nmol lipid/mg protein/30 min) when compared to liver (P < 0.05). 3. The inverse was true for liver homogenates when calculated for tissue activity (nmol lipid/g tissue/30 min). 4. Lysophosphatidate was produced in greater (P < 0.05) amounts than all other glycerolipids in the glycerol-3-phosphate acyltransferase assay. 5. The activity of phosphatidate phosphohydrolase in liver homogenates displayed greater rates than their respective adipose tissue homogenates. 6. Diacylglycerol acyltransferase activity was greater in adipose tissue homogenates compared to liver homogenates.     

Triacylglycerol biosynthesis in everted sacs of rat intestinal mucosa.

The molecular specificity of the biosynthesis of triacylglycerols by rat intestinal mucosa was examined by means of radioactive and mass tracers, and thin-layer chromatography with silver nitrate and gas-liquid chromatography with radioactivity monitoring. Bile salt micelles of alternately labeled monoacylglycerols and free fatty acids were incubated with everted sacs of intestinal mucosa for various periods of time and the triacylglycerols isolated by solvent extraction and thin-layer chromatography. Analyses of the molecular species of the triacylglycerols labeled from monoacylglycerols showed that the 2-monoacylglycerol pathway was responsible for the biosynthesis of a maximum of 90% and the X-1-monoacylglycerol pathway for about 10% of the total radioactive triacylglycerols. Detailed analyses of the molecular species of triacylglycerols labeled fro free fatty acids showed that the phosphatidic acid pathway contributed a minimum of 20-30% of the total labeled triacylglycerol formed. There was a preferential utilization in triacylglycerol biosynthesis of the more unsaturated diacylglycerols arising from the monoacylglycerol pathway and of the more saturated diacylglycerols originating from the phosphatidic acid pathway. The above experiments do not allow a demonstration of the utilization of the sn-2,3-diacylglycerols in triacylglycerol biosynthesis but are not inconsistent with it.     

Triacylglycerol biosynthesis in the adipose tissue of the obese-hyperglycaemic mouse.

Obesity in obese-hyperglycaemic mouse is associated with an increase in number and size of adipocytes. Adipocytes from the obese mouse showed increased incorporation of [14C]acetate and[14C]glucose into triacylglycerol. This increased capacity of triacylglycerol formation was correlated with increased activities of various triacylglycerol-forming enzymes measured in the microsomal fraction of adipose tissue from obese mice. Microsomal fractions from lean and obese mice contained sn-glycerol 3-phosphate acyltransferase, phosphatidate phosphohydrolase and diacylglycerol acyltransferase. Phosphatidate phosphohydrolase was also detected in the soluble fraction. In the presence of Mg2+, the phosphatidate phsophohydrolase from the soluble and the microsomal fractions was active towards membrane-bound phosphatidate. Among the three enzymes studied here, the increase in Mg2+-dependent phosphatidate phosphohydrolase was most prominent in adipose tissue of obese mice.     

Triacylglycerol synthesis in lipid particles from baker's yeast (Saccharomyces cerevisiae).

Triacylglycerol synthesis has been studied in a lipid particle preparation of baker's yeast (Saccharomyces cerevisiae), and compared with the synthesis in other subcellular fractions. Fatty acid-CoA ligase (AMP) (EC 6.2.1.3) activity and sn-glycerol 3-phosphate acyltransferase activity (EC 2.3.1.15) were present in all the subcellular fractions tested but the highest specific activities of both enzymes were observed with the lipid particle fraction. The products of the glycerol 3-phosphate acylation indicate that triacyglycerol synthesis proceeds through the phosphatidic acid pathway. However, only a small and nearly constant amount of lysophosphatidic acid was found with the lipid particle fraction while the other subcellular fraction produced lysophosphatidic and phosphatidic acid with a more pronounced precursor/product relationship. Triacylglycerol synthesis from endogenous diacylglycerol present in the lipid particle was also demonstrated.     

Identification of rate-limiting steps in yeast heme biosynthesis

The heme biosynthesis pathway in the yeast Saccharomyces cerevisiae is a highly regulated system, but the mechanisms accounting for this regulation remain unknown. In an attempt to identify rate-limiting steps in heme synthesis, which may constitute potential regulatory points, we constructed yeast strains overproducing two enzymes of the pathway: the porphobilinogen synthase (PBG-S) and deaminase (PBG-D). Biochemical analysis of the enzyme-overproducing strains revealed intracellular porphobilinogen and porphyrin accumulation. These results indicate that both enzymes play a rate-limiting role in yeast heme biosynthesis.     

Triacylglycerol biosynthesis in developing seeds of Tropaeolum majus L. and Limnanthes douglasii R. Br.

Triacylglycerols of both Tropaeolum majus L. and Limnanthes douglasii R. Br. are predominantly esterified with very long-chain acyl groups at each position of the glycerol backbone. In order to elucidate whether these acyl groups are directly chanelled into the triacylglycerols via the stepwise acylation of glycerol-3-phosphate, seed oil formation has been investigated in developing embryos of both plant species. [1-¹⁴C]Acetate labelling experiments using embryos at different stages of development, as well as the determination of the properties of the microsomal acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) and acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase (EC 2.3.1.51), revealed differences between the two plant species, especially with respect to the incorporation of very longchain acyl groups into the C2 position of the triacylglycerols. In microsomal fractions of developing embryos of L. douglasii both a glycerol-3-phosphate and a 1-acylglycerol-3-phosphate acyltransferase were detected which utilize very long-chain acyl-CoA thioesters as substrates. Thus, in seeds of L. douglasii very long-chain acyl groups can enter not only the C1, but also the C2 position of the triacylglycerols in the course of de-novo biosynthesis. A comparison of the properties of the acyltransferases of developing embryos with those of the corresponding activities of leaves indicates an embryo specific expression of an erucoyl-CoA-dependent microsomal 1-acylglycerol-3-phosphate acyltransferase in L. douglasii. The microsomal glycerol-3-phosphate acyltransferase of developing embryos of T. majus displayed properties very similar to those of the corresponding activity of L. douglasii. On the other hand, the microsomal 1-acylglycerol-3-phosphate acyltransferases of the two plant species showed strikingly different substrate specificities. Irrespective of the acyl groups of 1-acylglycerol-3-phosphate and regardless of whether acyl-CoA thioesters were offered separately or in mixtures, the enzyme of T. majus, in contrast to that of L. douglasii, was inactive with erucoyl-CoA. These results of the enzyme studies correspond well with those of the [1-¹⁴C]acetate labelling experiments and thus indicate that T. majus has developed mechanisms different from those of L. douglasii for the incorporation of erucic acid into the C2 position of its triacylglycerols.Abbreviations GPAT acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) - LPAT acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase (EC 2.3.1.51) This work was supported by the Bundesministerium für Forschung und Technologie (Förderkennzeichen 0316600A).     

Homoazasterol-mediated inhibition of yeast sterol biosynthesis.

A naturally occurring azasterol has been shown to inhibit sterol transmethylation in both in vitro and in vivo in the yeast Saccharomyces cerevisiae. The inhibition was competitive, with a calculated dissociation constant of 43 muM. The compound prevented the accumulation of ergosterol in aerobically adapting cells. Cultures forced to gain energy by respiration were found to be much more sensitive to growth inhibition by the azasterol than those cells fermenting glucose. The growth inhibition is reversible at low concentrations of the azasterol.     

Triacylglycerol lipase in bovine erythrocytes

Acid lipase activity was found in the bovine erythrocyte ghosts, but little neutral or alkaline lipase activity was observed in the erythrocytes. The membrane-bound lipase showed a remarkable activity in the ghosts only after hemolysis. The membrane-bound lipase showed its maximum activity at pH 4.5, 38 degrees C, and it was stable below 40 degrees C. The hydrolysis rate was linear with time up to 60 min, and was proportional to the amount of enzyme up to 0.4 mg protein. The bound lipase was activated markedly by bovine serum albumin and slightly by octyl-glycoside. The lipase was remarkably inhibited by bovine serum.