Phosphatidate phosphatase (PAP) catalyzes the conversion of phosphatidate (PA) to diacylglycerol (DAG) in a reaction that depends on Mg2+. This reaction is critical for de novo lipid accumulation because it provides the DAG needed for the biosynthesis of triacylglycerol (TAG). Lipid accumulation is triggered by the downregulation of the citric acid cycle, which results in the exit of citrate from the mitochondria to the cytosol. In the cytosol, citrate is converted to oxaloacetate and acetyl-CoA which is channeled to fatty acid biosynthesis. Oxaloacetate is converted to malate by malate dehydrogenase and malate to pyruvate by malic enzyme. Also, citrate stimulates the activity of acetyl-CoA carboxylase, which catalyzes the rate-limiting step in fatty acid biosynthesis. In this work, we examined the effect of citric acid cycle metabolites on PAP activity in cell extracts prepared from wild type cells and cells that lack PAH1 (i.e., pah1Δ). The cells were grown for 96 h on high glycerol media that induce lipid accumulation, and cell extracts were prepared. Control PAP assays done in the presence of Mg2+ showed that the pah1Δ mutation resulted in a 95% decrease in PAP activity, indicating that PAH1 encoded for almost all PAP activity. The effects of citrate, malate, and pyruvate were examined at concentrations ranging from 0.1 mM to 5 mM. In wild type cell extracts, citrate (1 mM), malate (0.1 mM), and pyruvate (2.5 mM) caused a 220%, 217%, and 154% increase in PAP activity, respectively. In contrast, the addition of these acids to cell extracts prepared from pah1Δ cells did not affect PAP activity. The stimulatory effect of these organic acids on PAP activity could provide a direct link between fatty acid biosynthesis and DAG synthesis by Pah1. The buildup of citrate and its metabolites in the cytosol could induce PAP activity to direct the lipid biosynthetic pathway towards the synthesis of DAG. This regulation, combined with the stimulation of acetyl-CoA carboxylase by citrate, could contribute to the induction in TAG synthesis typically observed during lipogenesis.
This work was supported by the Capacity Building Grants Program Grant 2020-38821-31118 from the USDA National Institute of Food and Agriculture.
