The Catalytic Activity of the PAH1‐encoded Phosphatidate Phosphatase is Required for Lipid Biosynthesis in the Oleaginous Yarrowia lipolytica

Abstract

The PAH1 gene encodes phosphatidate phosphatase (PAP) which catalyzes the conversion of phosphatidate (PA) to diacylglycerol used for the synthesis of triacylglycerol (TAG). Previous research in our lab showed that the lack of Pah1 (i.e. pah1Δ) in Y. lipolytica causes a significant decrease in cellular lipid content and TAG levels, while the phospholipid levels are increased. However, it is unclear whether this phenotype is due to the loss of the catalytic activity of Pah1 or due to some other function of the protein (e.g. transcriptional regulation). The objective of this work was to confirm that the loss of the catalytic activity of Pah1 is responsible for the phenotypes presented by pah1Δ cells.Yarrowia lipolytica pah1Δ cells expressing either wild‐type Pah1 fused with Green fluorescent protein (GFP) or a catalytically inactive Pah1 (i.e., D350E) fused with GFP were grown in media that favor lipid biosynthesis. PAP activity was measured following the release of water‐soluble 32Pi from chloroform‐soluble [32P] PA, while lipid profiles were analyzed by thin layer chromatography. The expression of the GFP fusions was confirmed by immunoblot analysis using antibodies directed against GFP. The results showed that the pah1Δ strain expressing an inactive Pah1 presented a decrease in TAG levels as well as overall lipid content and an increase in phospholipids compared to the pah1Δ strain expressing the wild‐type Pah1. These results showed that the catalytic activity of Pah1 is required for TAG biosynthesis in Y. lipolytica.Support or Funding InformationThis work was supported, in whole or in part, by National Institutes of Health Grant R15GM124602‐01 from the United States Public Health Service. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.