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The PHO4 Transcription Factor Regulates Triacylglycerol Metabolism under Low Phosphate Conditions in Saccharomyces cerevisiae
Author(s) -
Yadav Kamlesh,
Singh Neelima,
Rajasekharan Ram
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.715.38
Subject(s) - biochemistry , phosphatase , electrophoretic mobility shift assay , microbiology and biotechnology , saccharomyces cerevisiae , activator (genetics) , chemistry , transcription factor , enzyme , lysophosphatidic acid , monoacylglycerol lipase , biology , gene , endocannabinoid system , receptor
Objective To study the transcriptional regulation of lipid metabolism in Saccharomyces cerevisiae under low phosphate (Pi) conditions. Methodology [ 14 C]Acetate labeling, confocal microscopy, gas chromatography, semi quantitative and quantitative PCR. Electromobility shift assay (EMSA) and β‐galactosidase assay. To validate the binding site‐directed mutagenesis was performed. Enzyme kinetics and characterization. Results The expression of PHM8 is high under low phosphate conditions, which encodes a LPA phosphatase and nucleotidase. In an electrophoretic mobility shift assay, Pho4p was shown to bind specifically in PHM8 promoter. Analyses of PHM8 mRNA, LPA phosphatase activity and triacylglycerol levels under low phosphate conditions in phm8Δ showed that the plasmid harbouring mutated promoter along with the PHM8 gene had much lower PHM8 expression than the plasmid harbouring the native promoter with the gene. Enzyme kinetics with the differentially phosphorylated Phm8p using LPA and CMP as substrates showed that LPA is the preferred over CMP under low phosphate conditions. The results indicate that the transcriptional activator Pho4p binds to the PHM8 promoter under low phosphate conditions, activating PHM8 expression and leading to the formation of monoacylglycerol, which is successively acylated by Dga1p to form diacylglycerol and triacylglycerol.Conclusion The phosphate‐mediated transcriptional activation of PHM8 by Pho4p affects triacylglycerol biosynthesis by controlling lysophosphatidic acid and MAG flux.