YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae.
Previous work from our laboratory (Athenstaedt, K., Zweytick, D., Jandrositz, A., Kohlwein, S. D., and Daum, G. (1999) J. Bacteriol. 181, 6441-6448) showed that the gene product of YMR313c (named Tgl3p) is a component of yeast lipid particles, and deletion of this gene led to an increase in the cellular level of triacylglycerols (TAG). These observations suggested that TGL3 may encode a TAG lipase of Saccharomyces cerevisiae. Here we demonstrate by cell fractionation and by microscopic inspection of a strain bearing a Tgl3p-GFP hybrid that this polypeptide is highly enriched in the lipid particle fraction but virtually absent from other organelles. The entire TAG lipase activity of lipid particles is attributed to Tgl3p, because the activity in this organelle is completely absent in a Deltatgl3 deletion mutant, whereas it is significantly enhanced in a strain overexpressing Tgl3p. A His6-tagged Tgl3p hybrid purified close to homogeneity from a yeast strain overexpressing this fusion protein exhibited high TAG lipase activity. Most importantly, experiments in vivo using the fatty acid synthesis inhibitor cerulenin demonstrated that deletion of TGL3 resulted in a decreased mobilization of TAG from lipid particles. The amino acid sequence deduced from the open reading frame YMR313c contains the consensus sequence motif GXSXG typical for lipolytic enzymes. Otherwise, Tgl3p has no significant sequence homology to other lipases identified so far. In summary, our data identified Tgl3p as a novel yeast TAG lipase at the molecular level and by function in vivo and in vitro.
Athenstaedt
Karin
K
Institut für Biochemie, Technische Universität Graz, Petersgasse 12/2, A-8010 Graz, Austria.
Daum
Gunther
G
D002458:Cell Fractionation; D016384:Consensus Sequence; D049452:Green Fluorescent Proteins; D008049:Lipase / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D008055:Lipids / Q000032:analysis; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D020539:Sequence Analysis, Protein; D017385:Sequence Homology
D008055:Lipids; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D008049:Lipase; C476342:TGL3 protein, S cerevisiae