The Experts below are selected from a list of 213 Experts worldwide ranked by ideXlab platform
Guenther Daum - One of the best experts on this subject based on the ideXlab platform.
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modifications of the c terminus affect functionality and stability of yeast Triacylglycerol Lipase tgl3p
Journal of Biological Chemistry, 2014Co-Authors: Barbara Koch, Claudia Schmidt, Birgit Ploier, Guenther DaumAbstract:Lipid droplets are specific organelles for the storage of Triacylglycerols and steryl esters. They are surrounded by a phospholipid monolayer with a small but specific set of proteins embedded. Assembly and insertion of proteins into this surface membrane is an intriguing question of lipid droplet biology. To address this question we studied the topology of Tgl3p, the major Triacylglycerol Lipase of the yeast Saccharomyces cerevisiae, on lipid droplets. Employing the method of limited proteolysis of lipid droplet surface proteins, we found that the C terminus of Tgl3p faces the inside of the organelle, whereas the N terminus is exposed at the cytosolic side of lipid droplets. Detailed analysis of the C terminus revealed a stretch of seven amino acids that are critical for protein stability and functionality. The negative charge of two aspartate residues within this stretch is crucial for Lipase activity of Tgl3p. A portion of Tgl3p, which is located to the endoplasmic reticulum, exhibits a different topology. In the phospholipid bilayer of the endoplasmic reticulum the C terminus faces the cytosol, which results in instability of the protein. Thus, the topology of Tgl3p is important for its function and strongly dependent on the membrane environment.
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screening for hydrolytic enzymes reveals ayr1p as a novel Triacylglycerol Lipase in saccharomyces cerevisiae
Journal of Biological Chemistry, 2013Co-Authors: Birgit Ploier, Claudia Schmidt, Barbara Koch, Melanie Scharwey, Jessica Schatte, Gerald N Rechberger, Manfred Kollroser, Albin Hermetter, Guenther DaumAbstract:Abstract Saccharomyces cerevisiae, as well as other eukaryotes, preserves free fatty acids and sterols in a biologically inert form, as Triacylglycerols and steryl esters. The major Triacylglycerol Lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p and Tgl5p (Athenstaedt et al., 2003, J. Biol. Chem 278, 23317-23323; Athenstaedt et al., 2005, J. Biol. Chem 280, 37301-37309). We observed that upon cultivation on oleic acid Triacylglycerol mobilization did not come to a halt in a yeast strain deficient of all currently known Triacylglycerol Lipases, indicating the presence of additional not yet characterized Lipases/esterases. Functional proteome analysis using Lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GxSxG Lipase motif, putative functions and subcellular localizations a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis and in vivo Triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as novel Triacylglycerol Lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results we discuss a possible link between lipid storage, lipid mobilization and peroxisomal utilization of fatty acids as carbon source.
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regulation of the yeast Triacylglycerol Lipase tgl3p by formation of nonpolar lipids
Journal of Biological Chemistry, 2013Co-Authors: Claudia Schmidt, Karin Athenstaedt, Barbara Koch, Birgit Ploier, Guenther DaumAbstract:Tgl3p, the major Triacylglycerol Lipase of the yeast Saccharomyces cerevisiae, is a component of lipid droplets but is also present in the endoplasmic reticulum in a minor amount. Recently, it was shown that this enzyme can also serve as a lysophospholipid acyltransferase (Rajakumari, S., and Daum, G. (2010) Mol. Biol. Cell 21, 501–510). Here, we describe the effects of the presence/absence of Triacylglycerols and lipid droplets on the functionality of Tgl3p. In a dga1Δlro1Δare1Δare2Δ quadruple mutant lacking all four Triacylglycerol- and steryl ester-synthesizing acyltransferases and consequently the lipid droplets, the gene expression of TGL3 was only slightly altered. In contrast, protein level and stability of Tgl3p were markedly reduced in the absence of lipid droplets. Under these conditions, the enzyme was localized to the endoplasmic reticulum. Even the lack of the substrate, Triacylglycerol, affected stability and localization of Tgl3p to some extent. Interestingly, Tgl3p present in the endoplasmic reticulum seems to lack lipolytic as well as acyltransferase activity as shown by enzymatic analysis and lipid profiling. Thus, we propose that the activity of Tgl3p is restricted to lipid droplets, whereas the endoplasmic reticulum may serve as a parking lot for this enzyme.
Peter J. Eastmond - One of the best experts on this subject based on the ideXlab platform.
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Triacylglycerol Lipase That Initiates Storage Oil Breakdown in Germinating Arabidopsis Seeds
2020Co-Authors: Peter J. EastmondAbstract:Triacylglycerol hydrolysis (lipolysis) plays a pivotal role in the life cycle of many plants by providing the carbon skeletons and energy that drive postgerminative growth. Despite the physiological importance of this process, the molecular mechanism is unknown. Here, a genetic screen has been used to identify Arabidopsis thaliana mutants that exhibit a postgerminative growth arrest phenotype, which can be rescued by providing sugar. Seventeen sugar-dependent (sdp) mutants were isolated, and six represent new loci. Triacylglycerol hydrolase assays showed that sdpl, sdp2, and sdp3 seedlings are deficient specifically in the Lipase activity that is associated with purified oil bodies. Map-based cloning of SDP1 revealed that it encodes a protein with a patatin-like acyl-hydrolase domain. SDP1 shares this domain with yeast Triacylglycerol Lipase 3 and human adipose triglyc?ride Lipase. In vitro assays confirmed that recombinant SDP1 hydrolyzes Triacylglycerols and diacylglycerols but not monoacylglycerols, phospholipids, galactolipids, or cholesterol esters. SDP1 is expressed predominantly in developing seeds, and a SDP1 -green fluorescent protein fusion was shown to associate with the oil body surface in vivo. These data shed light on the mechanism of lipolysis in plants and establish that a central component is evolutionary conserved among eukaryotes.
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suppression of the sugar dependent1 Triacylglycerol Lipase family during seed development enhances oil yield in oilseed rape brassica napus l
Plant Biotechnology Journal, 2013Co-Authors: Amelie A Kelly, Eve Shaw, Stephen J Powers, Smita Kurup, Peter J. EastmondAbstract:Increasing the productivity of oilseed crops is an important challenge for plant breeders and biotechnologists. To date, attempts to increase oil production in seeds via metabolic pathway engineering have focused on boosting synthetic capacity. However, in the tissues of many organisms, it is well established that oil levels are determined by both anabolism and catabolism. Indeed, the oil content of rapeseed (Brassica napus L.) has been reported to decline by approximately 10% in the final stage of development, as the seeds desiccate. Here, we show that RNAi suppression of the SUGAR-DEPENDENT1 Triacylglycerol Lipase gene family during seed development results in up to an 8% gain in oil yield on either a seed, plant or unit area basis in the greenhouse, with very little adverse impact on seed vigour. Suppression of lipolysis could therefore constitute a new method for enhancing oil yield in oilseed crops.
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sugar dependent1 encodes a patatin domain Triacylglycerol Lipase that initiates storage oil breakdown in germinating arabidopsis seeds
The Plant Cell, 2006Co-Authors: Peter J. EastmondAbstract:Triacylglycerol hydrolysis (lipolysis) plays a pivotal role in the life cycle of many plants by providing the carbon skeletons and energy that drive postgerminative growth. Despite the physiological importance of this process, the molecular mechanism is unknown. Here, a genetic screen has been used to identify Arabidopsis thaliana mutants that exhibit a postgerminative growth arrest phenotype, which can be rescued by providing sugar. Seventeen sugar-dependent (sdp) mutants were isolated, and six represent new loci. Triacylglycerol hydrolase assays showed that sdp1, sdp2, and sdp3 seedlings are deficient specifically in the Lipase activity that is associated with purified oil bodies. Map-based cloning of SDP1 revealed that it encodes a protein with a patatin-like acyl-hydrolase domain. SDP1 shares this domain with yeast Triacylglycerol Lipase 3 and human adipose triglyceride Lipase. In vitro assays confirmed that recombinant SDP1 hydrolyzes Triacylglycerols and diacylglycerols but not monoacylglycerols, phospholipids, galactolipids, or cholesterol esters. SDP1 is expressed predominantly in developing seeds, and a SDP1–green fluorescent protein fusion was shown to associate with the oil body surface in vivo. These data shed light on the mechanism of lipolysis in plants and establish that a central component is evolutionarily conserved among eukaryotes.
Birgit Ploier - One of the best experts on this subject based on the ideXlab platform.
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modifications of the c terminus affect functionality and stability of yeast Triacylglycerol Lipase tgl3p
Journal of Biological Chemistry, 2014Co-Authors: Barbara Koch, Claudia Schmidt, Birgit Ploier, Guenther DaumAbstract:Lipid droplets are specific organelles for the storage of Triacylglycerols and steryl esters. They are surrounded by a phospholipid monolayer with a small but specific set of proteins embedded. Assembly and insertion of proteins into this surface membrane is an intriguing question of lipid droplet biology. To address this question we studied the topology of Tgl3p, the major Triacylglycerol Lipase of the yeast Saccharomyces cerevisiae, on lipid droplets. Employing the method of limited proteolysis of lipid droplet surface proteins, we found that the C terminus of Tgl3p faces the inside of the organelle, whereas the N terminus is exposed at the cytosolic side of lipid droplets. Detailed analysis of the C terminus revealed a stretch of seven amino acids that are critical for protein stability and functionality. The negative charge of two aspartate residues within this stretch is crucial for Lipase activity of Tgl3p. A portion of Tgl3p, which is located to the endoplasmic reticulum, exhibits a different topology. In the phospholipid bilayer of the endoplasmic reticulum the C terminus faces the cytosol, which results in instability of the protein. Thus, the topology of Tgl3p is important for its function and strongly dependent on the membrane environment.
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screening for hydrolytic enzymes reveals ayr1p as a novel Triacylglycerol Lipase in saccharomyces cerevisiae
Journal of Biological Chemistry, 2013Co-Authors: Birgit Ploier, Claudia Schmidt, Barbara Koch, Melanie Scharwey, Jessica Schatte, Gerald N Rechberger, Manfred Kollroser, Albin Hermetter, Guenther DaumAbstract:Abstract Saccharomyces cerevisiae, as well as other eukaryotes, preserves free fatty acids and sterols in a biologically inert form, as Triacylglycerols and steryl esters. The major Triacylglycerol Lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p and Tgl5p (Athenstaedt et al., 2003, J. Biol. Chem 278, 23317-23323; Athenstaedt et al., 2005, J. Biol. Chem 280, 37301-37309). We observed that upon cultivation on oleic acid Triacylglycerol mobilization did not come to a halt in a yeast strain deficient of all currently known Triacylglycerol Lipases, indicating the presence of additional not yet characterized Lipases/esterases. Functional proteome analysis using Lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GxSxG Lipase motif, putative functions and subcellular localizations a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis and in vivo Triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as novel Triacylglycerol Lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results we discuss a possible link between lipid storage, lipid mobilization and peroxisomal utilization of fatty acids as carbon source.
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regulation of the yeast Triacylglycerol Lipase tgl3p by formation of nonpolar lipids
Journal of Biological Chemistry, 2013Co-Authors: Claudia Schmidt, Karin Athenstaedt, Barbara Koch, Birgit Ploier, Guenther DaumAbstract:Tgl3p, the major Triacylglycerol Lipase of the yeast Saccharomyces cerevisiae, is a component of lipid droplets but is also present in the endoplasmic reticulum in a minor amount. Recently, it was shown that this enzyme can also serve as a lysophospholipid acyltransferase (Rajakumari, S., and Daum, G. (2010) Mol. Biol. Cell 21, 501–510). Here, we describe the effects of the presence/absence of Triacylglycerols and lipid droplets on the functionality of Tgl3p. In a dga1Δlro1Δare1Δare2Δ quadruple mutant lacking all four Triacylglycerol- and steryl ester-synthesizing acyltransferases and consequently the lipid droplets, the gene expression of TGL3 was only slightly altered. In contrast, protein level and stability of Tgl3p were markedly reduced in the absence of lipid droplets. Under these conditions, the enzyme was localized to the endoplasmic reticulum. Even the lack of the substrate, Triacylglycerol, affected stability and localization of Tgl3p to some extent. Interestingly, Tgl3p present in the endoplasmic reticulum seems to lack lipolytic as well as acyltransferase activity as shown by enzymatic analysis and lipid profiling. Thus, we propose that the activity of Tgl3p is restricted to lipid droplets, whereas the endoplasmic reticulum may serve as a parking lot for this enzyme.
Claudia Schmidt - One of the best experts on this subject based on the ideXlab platform.
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modifications of the c terminus affect functionality and stability of yeast Triacylglycerol Lipase tgl3p
Journal of Biological Chemistry, 2014Co-Authors: Barbara Koch, Claudia Schmidt, Birgit Ploier, Guenther DaumAbstract:Lipid droplets are specific organelles for the storage of Triacylglycerols and steryl esters. They are surrounded by a phospholipid monolayer with a small but specific set of proteins embedded. Assembly and insertion of proteins into this surface membrane is an intriguing question of lipid droplet biology. To address this question we studied the topology of Tgl3p, the major Triacylglycerol Lipase of the yeast Saccharomyces cerevisiae, on lipid droplets. Employing the method of limited proteolysis of lipid droplet surface proteins, we found that the C terminus of Tgl3p faces the inside of the organelle, whereas the N terminus is exposed at the cytosolic side of lipid droplets. Detailed analysis of the C terminus revealed a stretch of seven amino acids that are critical for protein stability and functionality. The negative charge of two aspartate residues within this stretch is crucial for Lipase activity of Tgl3p. A portion of Tgl3p, which is located to the endoplasmic reticulum, exhibits a different topology. In the phospholipid bilayer of the endoplasmic reticulum the C terminus faces the cytosol, which results in instability of the protein. Thus, the topology of Tgl3p is important for its function and strongly dependent on the membrane environment.
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screening for hydrolytic enzymes reveals ayr1p as a novel Triacylglycerol Lipase in saccharomyces cerevisiae
Journal of Biological Chemistry, 2013Co-Authors: Birgit Ploier, Claudia Schmidt, Barbara Koch, Melanie Scharwey, Jessica Schatte, Gerald N Rechberger, Manfred Kollroser, Albin Hermetter, Guenther DaumAbstract:Abstract Saccharomyces cerevisiae, as well as other eukaryotes, preserves free fatty acids and sterols in a biologically inert form, as Triacylglycerols and steryl esters. The major Triacylglycerol Lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p and Tgl5p (Athenstaedt et al., 2003, J. Biol. Chem 278, 23317-23323; Athenstaedt et al., 2005, J. Biol. Chem 280, 37301-37309). We observed that upon cultivation on oleic acid Triacylglycerol mobilization did not come to a halt in a yeast strain deficient of all currently known Triacylglycerol Lipases, indicating the presence of additional not yet characterized Lipases/esterases. Functional proteome analysis using Lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GxSxG Lipase motif, putative functions and subcellular localizations a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis and in vivo Triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as novel Triacylglycerol Lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results we discuss a possible link between lipid storage, lipid mobilization and peroxisomal utilization of fatty acids as carbon source.
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regulation of the yeast Triacylglycerol Lipase tgl3p by formation of nonpolar lipids
Journal of Biological Chemistry, 2013Co-Authors: Claudia Schmidt, Karin Athenstaedt, Barbara Koch, Birgit Ploier, Guenther DaumAbstract:Tgl3p, the major Triacylglycerol Lipase of the yeast Saccharomyces cerevisiae, is a component of lipid droplets but is also present in the endoplasmic reticulum in a minor amount. Recently, it was shown that this enzyme can also serve as a lysophospholipid acyltransferase (Rajakumari, S., and Daum, G. (2010) Mol. Biol. Cell 21, 501–510). Here, we describe the effects of the presence/absence of Triacylglycerols and lipid droplets on the functionality of Tgl3p. In a dga1Δlro1Δare1Δare2Δ quadruple mutant lacking all four Triacylglycerol- and steryl ester-synthesizing acyltransferases and consequently the lipid droplets, the gene expression of TGL3 was only slightly altered. In contrast, protein level and stability of Tgl3p were markedly reduced in the absence of lipid droplets. Under these conditions, the enzyme was localized to the endoplasmic reticulum. Even the lack of the substrate, Triacylglycerol, affected stability and localization of Tgl3p to some extent. Interestingly, Tgl3p present in the endoplasmic reticulum seems to lack lipolytic as well as acyltransferase activity as shown by enzymatic analysis and lipid profiling. Thus, we propose that the activity of Tgl3p is restricted to lipid droplets, whereas the endoplasmic reticulum may serve as a parking lot for this enzyme.
Barbara Koch - One of the best experts on this subject based on the ideXlab platform.
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modifications of the c terminus affect functionality and stability of yeast Triacylglycerol Lipase tgl3p
Journal of Biological Chemistry, 2014Co-Authors: Barbara Koch, Claudia Schmidt, Birgit Ploier, Guenther DaumAbstract:Lipid droplets are specific organelles for the storage of Triacylglycerols and steryl esters. They are surrounded by a phospholipid monolayer with a small but specific set of proteins embedded. Assembly and insertion of proteins into this surface membrane is an intriguing question of lipid droplet biology. To address this question we studied the topology of Tgl3p, the major Triacylglycerol Lipase of the yeast Saccharomyces cerevisiae, on lipid droplets. Employing the method of limited proteolysis of lipid droplet surface proteins, we found that the C terminus of Tgl3p faces the inside of the organelle, whereas the N terminus is exposed at the cytosolic side of lipid droplets. Detailed analysis of the C terminus revealed a stretch of seven amino acids that are critical for protein stability and functionality. The negative charge of two aspartate residues within this stretch is crucial for Lipase activity of Tgl3p. A portion of Tgl3p, which is located to the endoplasmic reticulum, exhibits a different topology. In the phospholipid bilayer of the endoplasmic reticulum the C terminus faces the cytosol, which results in instability of the protein. Thus, the topology of Tgl3p is important for its function and strongly dependent on the membrane environment.
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screening for hydrolytic enzymes reveals ayr1p as a novel Triacylglycerol Lipase in saccharomyces cerevisiae
Journal of Biological Chemistry, 2013Co-Authors: Birgit Ploier, Claudia Schmidt, Barbara Koch, Melanie Scharwey, Jessica Schatte, Gerald N Rechberger, Manfred Kollroser, Albin Hermetter, Guenther DaumAbstract:Abstract Saccharomyces cerevisiae, as well as other eukaryotes, preserves free fatty acids and sterols in a biologically inert form, as Triacylglycerols and steryl esters. The major Triacylglycerol Lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p and Tgl5p (Athenstaedt et al., 2003, J. Biol. Chem 278, 23317-23323; Athenstaedt et al., 2005, J. Biol. Chem 280, 37301-37309). We observed that upon cultivation on oleic acid Triacylglycerol mobilization did not come to a halt in a yeast strain deficient of all currently known Triacylglycerol Lipases, indicating the presence of additional not yet characterized Lipases/esterases. Functional proteome analysis using Lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GxSxG Lipase motif, putative functions and subcellular localizations a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis and in vivo Triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as novel Triacylglycerol Lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results we discuss a possible link between lipid storage, lipid mobilization and peroxisomal utilization of fatty acids as carbon source.
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regulation of the yeast Triacylglycerol Lipase tgl3p by formation of nonpolar lipids
Journal of Biological Chemistry, 2013Co-Authors: Claudia Schmidt, Karin Athenstaedt, Barbara Koch, Birgit Ploier, Guenther DaumAbstract:Tgl3p, the major Triacylglycerol Lipase of the yeast Saccharomyces cerevisiae, is a component of lipid droplets but is also present in the endoplasmic reticulum in a minor amount. Recently, it was shown that this enzyme can also serve as a lysophospholipid acyltransferase (Rajakumari, S., and Daum, G. (2010) Mol. Biol. Cell 21, 501–510). Here, we describe the effects of the presence/absence of Triacylglycerols and lipid droplets on the functionality of Tgl3p. In a dga1Δlro1Δare1Δare2Δ quadruple mutant lacking all four Triacylglycerol- and steryl ester-synthesizing acyltransferases and consequently the lipid droplets, the gene expression of TGL3 was only slightly altered. In contrast, protein level and stability of Tgl3p were markedly reduced in the absence of lipid droplets. Under these conditions, the enzyme was localized to the endoplasmic reticulum. Even the lack of the substrate, Triacylglycerol, affected stability and localization of Tgl3p to some extent. Interestingly, Tgl3p present in the endoplasmic reticulum seems to lack lipolytic as well as acyltransferase activity as shown by enzymatic analysis and lipid profiling. Thus, we propose that the activity of Tgl3p is restricted to lipid droplets, whereas the endoplasmic reticulum may serve as a parking lot for this enzyme.
