PHO4 transcription factor regulates triacylglycerol metabolism under low‐phosphate conditions in Saccharomyces cerevisiae

Abstract

SummaryIn Saccharomyces cerevisiae, PHM8 encodes a phosphatase that catalyses the dephosphorylation of lysophosphatidic acids to monoacylglycerol and nucleotide monophosphate to nucleoside and releases free phosphate. In this report, we investigated the role of PHM8 in triacylglycerol metabolism and its transcriptional regulation by a phosphate responsive transcription factor Pho4p under lowphosphate conditions. We found that the wild-type (BY4741) cells accumulate triacylglycerol and the expression of PHM8 was high under low-phosphate conditions. Overexpression of PHM8 in the wildtype, phm8Δ and quadruple phosphatase mutant (pah1Δdpp1Δlpp1Δapp1Δ) caused an increase in the triacylglycerol levels. However, the introduction of the PHM8 deletion into the quadruple phosphatase mutant resulted in a reduction in triacylglycerol levels and LPA phosphatase activity. The transcriptional activator Pho4p binds to the PHM8 promoter under low-phosphate conditions, activating PHM8 expression, which leads to the formation of monoacylglycerol from LPA. The synthesized monoacylglycerol is acylated to diacylglycerol by Dga1p, which is further acylated to triacylglycerol by the same enzyme.

MGL2/YMR210w encodes a monoacylglycerol lipase in Saccharomyces cerevisiae

“…After centrifugation (1344 g at 4 °C for 30 min), the soluble fraction was loaded onto a Ni 2+ –NTA column and eluted using the buffer containing 50 m m Tris‐HCl (pH 8.0), 150 m m NaCl, 1% glycerol, and 250 m m imidazole. For isolating the Mgl2p from membranes, the membrane fraction was solubilized in the lysis buffer containing 1% dodecyl β‐ d maltoside (DDM) at 4 °C for 60 min and centrifuged at 1 50 000 g for 60 min . The solubilized membrane fraction was then loaded on to Ni 2+ –nitrilotriacetic acid column chromatography, and the protein was eluted with the elution buffer (50 m m Tris, 150 m m NaCl, 250 m m Imidazole and 0.1% DDM).…”

ATG15 encodes a phospholipase and is transcriptionally regulated by YAP1 in Saccharomyces cerevisiae

“…The assay was carried out in a binding buffer containing 20 m m HEPES (pH 7.9), 50 m m NaCl, 1 m m EDTA, 5% glycerol, 5 m m MgCl 2 , and 1 m m dithiothreitol, and the binding mixture was incubated at 30 °C for 1 h. The resulting DNA–protein complexes were resolved on a 6% nondenaturing polyacrylamide gel and electrophoresed at 50 V at 4 °C. The gel was stained with SYBR green EMSA nucleic acid gel stain (Life Technologies) for 1 h, and the bands were visualized under UV illumination .…”

Cardiolipin deficiency causes triacylglycerol accumulation in Saccharomyces cerevisiae

“…9; Building N250, D-60438 Frankfurt, Germany). The pYES2/NT B-DDL1 (pRP1) and pYES2/NT C-DGA1 (pRK13) plasmids were described previously [18,19]. The yeast strains used in this work were stored as glycerol stocks at -80°C.…”

“…The obtained control value (mean, n = 3) for the RF was set to 100%. pRK13 DGA1 gene is cloned into pYES2/NT C: pYES2/NT C-DGA1 Yadav et al [19] Real-time quantitative PCR analysis Cells (A 600 = 25) from the stationary phase were harvested and stored in RNAlater at -20°C. Total RNA isolation was performed using an RNA isolation kit (NucleoSpinÒ RNA II kit, Macherey-Nagel), and a high-capacity cDNA reverse transcription kit (Applied Biosystems) was used for the complementary DNA (cDNAs) synthesis.…”

“…Glass beads were used for the cell lysis. Microsomal membranes were prepared and solubilized with 20 mM n-dodecyl b-D-maltoside (DDM) detergent according to the procedure used by Yadav et al [19]. Histagged Dga1 protein was affinity purified with the help of Ni 2?…”