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.     

Properties of triacylglycerol lipase in a mitochondrial fraction from baker's yeast (Saccharomyces cerevisiae).

A triacylglycerol lipase in a mitochondrial fraction isolated from yeast (Saccharomyces cerevisiae) has been characterized and the hydrolysis studied kinetically using an insoluble artificial triacylglycerol suspension. 1. The triacylglycerol was hydrolyzed almost completely to fatty acids and glycerol. The lipase activity was inhibited by potassium fluoride and the sodium salts of -chloride, -glycocholate and -pyrophosphate as well as by protamine sulfate but at concentrations much too high to indicate that the lipase is a non specific esterase or a lipoprotein lipase. Also parachloromercuribenzoate inhibited the lipase activity. Inhibitory effect of fatty acid was observed at concentrations above 1mM. This inhibition may provide a regulatory mechanism of the lipase in vivo. 2. On the day of isolation the lipase activity of intact mitochondria at pH 7.5 and 30 degrees C was 400 nmol free fatty acid -h-1 - mg-1 at a triacylglycerol concentration of 9.0 mM. Sonication of the mitochondria increased the activity 2-3 fold. Freezing of the mitochondria also activated the lipase and this activation was dependent upon the freezing method, the concentration of mitochondrial protein and the presence of bovine serum albumin. 3. The particulate nature of the assay system was illustrated by the observation that the apparent Km value of the lipase increased with the concentration of mitochondrial protein. For each protein concentration the lipase had two apparent Km values when the activity was assayed with intact mitochondria, but only one when assayed with submitochondrial particles. At the same protein concentration the Km value for the latter was identical with the "low affinity" Km for the lipase in intact mitochondria.     

Purification and properties of tryptophan synthase from baker's yeast (Saccharomyces cerevisiae).

Tryptophan synthase was purified from baker's yeast. The purified enzyme exhibited one band on polyacrylamide-gel electrophoresis, had no detectable N-terminal amino acid and C-terminal alanine. The amino acid composition was close to that predicted by recent studies on the DNA sequence of the structural gene for the enzyme. Kinetic parameters for the following three activities were measured: indole-serine condensation, indolylglycerol phosphate lyase and the overall reaction of serine with 1-(indol-3-yl)glycerol 3-phosphate. The Km for indole was much lower than suggested by previous investigations, and the value of 11 microM was measured by a fluorimetric assay.     

Circular dichroism studies on cytochrome c peroxidase from baker's yeast (Saccharomyces cerevisiae).

Circular dichroism spectra of cytochrome c peroxidase from baker's yeast, those of the reduced enzyme, the carbonyl, cyanide and fluoride derivatives and the hydrogen peroxide compound, Compound I, have been recorded in the wavelength range 200 to 660 nm. All derivatives show negative Soret Cotton effects. The results suggest that the heme group is surrounded by tightly packed amino acid sidechains and that there is a histidine residue bound to the fifth coordination site of the heme iron. The native ferric enzyme is probably pentacoordinated. The circular dichroism spectra of the ligand compounds indicate that the ligands form a nonlinear bond to the heme iron as a result of steric hindrance in the vicinity of the heme. The spectrum of Compound I shows no perturbation of the porphyrin symmetry. The dichroic spectrum of the native enzyme in the far-ultraviolet wave-length region suggests that the secondary structure consists of roughly equal amounts of alpha-helical, beta-structure and unordered structure. After the removal of the heme group no great changes in the secondary structure can be observed.     

Structural and biochemical properties of lipid particles from the yeast Saccharomyces cerevisiae

The two most prominent neutral lipids of the yeast Saccharomyces cerevisiae, triacylglycerols (TAG) and steryl esters (SE), are synthesized by the two TAG synthases Dga1p and Lro1p and the two SE synthases Are1p and Are2p. In this study, we made use of a set of triple mutants with only one of these acyltransferases active to elucidate the contribution of each single enzyme to lipid particle (LP)/droplet formation. Depending on the remaining acyltransferases, LP from triple mutants contained only TAG or SE, respectively, with specific patterns of fatty acids and sterols. Biophysical investigations, however, revealed that individual neutral lipids strongly affected the internal structure of LP. SE form several ordered shells below the surface phospholipid monolayer of LP, whereas TAG are more or less randomly packed in the center of the LP. We propose that this structural arrangement of neutral lipids in LP may be important for their physiological role especially with respect to mobilization of TAG and SE reserves.     

Activation of serine sulphydrase from baker's yeast (Saccharomyces cerevisiae) by vanadate

Vanadate stimulates the liberation of H2S from cysteine in intact cells of baker's yeast (Saccharomyces cerevisiae) with a maximal increase of 60% at 10 microM NH4VO3. Protein separation from crude yeast extract yielded two active protein fractions which were found to catalyze the degradation of cysteine to H2S, pyruvate and ammonia or H2S and serine, respectively, thus characterizing them as cysteine desulphydrase and serine sulphydrase. Only the latter enzyme was found to be activated by vanadate, showing optimal enhancement of about 100% at 10 microM NH4VO3.     

The three zinc-containing alcohol dehydrogenases from baker's yeast,Saccharomyces cerevisiae

This review is a summary of our current knowledge of the structure, function and mechanism of action of the three zinc-containing alcohol dehydrogenases, YADH-1, YADH-2 and YADH-3, in baker's yeast, Saccharomyces cerevisiae. The opening section deals with the substrate specificity of the enzymes, covering the steady-state kinetic data for its most known substrates. In the following sections, the kinetic mechanism for this enzyme is reported, along with the values of all rate constants in the mechanism. The complete primary structures of the three isoenzymes of YADH are given, and the model of the 3D structure of the active site is presented. All known artificial mutations in the primary structure of the YADH are covered in full and described in detail. Further, the chemical mechanism of action for YADH is presented along with the complement of steady-state and ligand-binding data supporting this mechanism. Finally, the bio-organic chemistry of the hydride-transfer reactions catalyzed by the enzyme is covered: this chemistry explains the narrow substrate specificity and the enantioselectivity of the yeast enzyme.     

Immunochemical analysis of the plasma membrane from baker's yeast Saccharomyces cerevisiae

Yeast plasma membranes have been isolated from homogenized yeast cells, identified as pure plasma membrane vesicles which were used as antigens. By crossed immunoelectrophoresis with anti-membrane immunoglobulins, 17 discrete antigens have been detected in Triton X-100 extracts from plasma membranes. Three different immunoabsorption experiments were performed with : a) isolated membranes exposing the cytoplasmic surfaces (PS) and the external surfaces (ES), b) yeast protoplasts exposing only antigenic determinants on the ES, c) lysed protoplasts which had been saturated on the ES with antibodies prior to lysis. These absorption experiments demonstrated that seven of the antigens are expressed on the ES while eight immunogens expose antigenic determinants on the PS. Four of the principal immunoprecipitates are not affected by absorption with surface antigens whereas two of the antigens indicate transmembrane characteristics. Of these 17 immunoprecipitates four were shown by zymograms to possess enzymatic activities: ATPase (EC 3.6.1.3) and NADH-dehydrogenase (EC 1.8.99.3) (three separate components). Three of these enzymes are expressed on the PS, and one NADH-dehydrogenase exposes determinants on the ES of the protoplasts. The presence of antigens on the PS of the plasma membrane could also be demonstrated on micrographs by the indirect ferritin-antibody labeling technique followed by freeze-etching and shadowing of the membranes.     

Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae are localized to lipid particles

Triacylglycerol (TAG) lipases are required for mobilization of TAG stored in lipid particles. Recently, Tgl3p was identified as a major TAG lipase of the yeast Saccharomyces cerevisiae (Athenstaedt, K., and Daum, G. (2003) J. Biol. Chem. 278, 23317-23323). Here, we report the identification of Tgl4p and Tgl5p as additional TAG lipases of the yeast. Both polypeptides, encoded by open reading frames YKR089c/TGL4 and YOR081c/TGL5, share 30 and 26% homology, respectively, to Tgl3p. Cell fractionation experiments and microscopic inspection of strains bearing Tgl4p-GFP and Tgl5p-GFP hybrids demonstrated that both proteins are localized to lipid particles similar to Tgl3p. A 1.7-fold increased amount of TAG enriched in myristic and palmitic acids and the reduced mobilization rate of TAG from tgl4Delta in the presence of the fatty acid synthesis inhibitor cerulenin demonstrated the lipolytic function of Tgl4p in vivo. In contrast, neither the total amount of TAG nor the TAG mobilization rate after addition of cerulenin was affected in tgl5Delta cells. However, the enrichment of C26:0 esterified to TAG of tgl5Delta, an additional increase of TAG in the tgl4Deltatgl5Delta double deletion mutant compared with tgl4Delta, and the impairment of TAG mobilization in the tgl4Deltatgl5Delta strain in the presence of cerulenin suggested that also Tgl5p functions as a TAG lipase in vivo. Most importantly, the purified His(6)-tagged Tgl4p and Tgl5p hybrids exhibited TAG lipase activity demonstrating their function in vitro. In summary, our data obtained by biochemical, molecular, and cell biological analyses unambiguously identified Tgl4p and Tgl5p as novel TAG lipases of yeast lipid particles with certain enzymatic specificities.     

The isolation and characterization of peroxisomes (microbodies) from baker's yeast,Saccharomyces cerevisiae

For anaerobic glucose-limited chemostat cultures of Aerobacter aerogenes a values of 14.0 g/mole was found for Y _ATP ^max and a value of 6.8 mmoles ATP/g dry weight/hr for the maintenance coefficient. Both values are much lower than those previously determined for tryptophan-limited anaerobic chemostat cultures. It is concluded that generally the largest part of the maintenance energy is not used for true maintenance processes. For aerobic glucose-limited chemostat cultures two phases could be differentiated. Acetate production started at μ values higher than 0.53. The slopes of the curves relating the specific rates of glucose- and oxygen consumption with μ became higher and lower respectively above the μ value of 0.53. Using the Y _ATP values obtained in the anaerobic experiment a P/O ratio of about 1.3 could be calculated for glucose- and tryptophan-limited chemostat cultures. In sulfate-limited chemostat cultures acetate was produced at all growth rates. At high growth rates also pyruvate and α-ketoglutarate were produced. With the Y _ATP values obtained in the anaerobic experiment a P/O ratio of about 0.4 was calculated for sulfate-limited chemostat cultures.     

Contribution of different pathways to the supply of phosphatidylethanolamine and phosphatidylcholine to mitochondrial membranes of the yeast Saccharomyces cerevisiae

In the yeast, three biosynthetic pathways lead to the formation of phosphatidylethanolamine (PtdEtn): (i) decarboxylation of phosphatidylserine (PtdSer) by phosphatidylserine decarboxylase 1 (Psd1p) in mitochondria; (ii) decarboxylation of PtdSer by Psd2p in a Golgi/vacuolar compartment; and (iii) the CDP-ethanolamine (CDP-Etn) branch of the Kennedy pathway. The major phospholipid of the yeast, phosphatidylcholine (PtdCho), is formed either by methylation of PtdEtn or via the CDP-choline branch of the Kennedy pathway. To study the contribution of these pathways to the supply of PtdEtn and PtdCho to mitochondrial membranes, labeling experiments in vivo with [(3)H]serine and [(14)C]ethanolamine, or with [(3)H]serine and [(14)C]choline, respectively, and subsequent cell fractionation were performed with psd1Delta and psd2Delta mutants. As shown by comparison of the labeling patterns of the different strains, the major source of cellular and mitochondrial PtdEtn is Psd1p. PtdEtn formed by Psd2p or the CDP-Etn pathway, however, can be imported into mitochondria, although with moderate efficiency. In contrast to mitochondria, microsomal PtdEtn is mainly derived from the CDP-Etn pathway. PtdEtn formed by Psd2p is the preferred substrate for PtdCho synthesis. PtdCho derived from the different pathways appears to be supplied to subcellular membranes from a single PtdCho pool. Thus, the different pathways of PtdEtn biosynthesis play different roles in the assembly of PtdEtn into cellular membranes.     

The presence of dolichol in a lipid diphosphate N-acetylglucosamine from Saccharomyces cerevisiae (baker''s yeast).

The lipid moiety of a lipid diphosphate N-acetylglucosamine, an intermediate in glycosylation of proteins, was studied. Ozonolysis of the compound gave evidence for an alpha-saturated isoprene unit. Alkaline hydrolysis of the glycolipid, followed by high-pressure liquid chromatography, showed the presence of a series of polyprenol homologues identical with those isolated directly from Saccharomyces cerevisiae (baker's yeast). No particular homologue was preferred in the enzymic transfer of N-acetylglucosamine 1-phosphate to endogenous dolichol monophosphate.     

Some aspects of catalase induction in baker's yeast (Saccharomyces cerevisiae)

Yeasts grown in anaerobic liquid media produced catalase in response to the presence of H₂O₂ in the growth medium. The fact that some of the induced enzyme was active at the cell surface, bound either to the cell wall or cell-surface membrane, eliminated the need to crush cells in order to release the enzyme complement. Instead, catalase production was monitored by using H₂O₂-reagent strips to detect changes in the level of H₂O₂, in the growth medium. In addition, catalase induction in yeasts was found to be temperature-sensitive. It is suggested that biology teachers in schools might find the following experiments useful for demonstrating essential features of substrate-induced enzyme synthesis, based on the Jacob-Monod model, and for showing that the activity of certain genes can be modified by environmental temperature.