Abstract 1485 Construction and analysis of the catalytic core of yeast Pah1 phosphatidate phosphatase

The PAH1-encoded phosphatidate (PA) phosphatase is a major source of diacylglycerol for the production of the storage lipid triacylglycerol, and a key regulator for the de novo phospholipid synthesis in Saccharomyces cerevisiae. The catalytic function of Pah1 depends on its membrane localization that is mediated through its phosphorylation by multiple protein kinases and dephosphorylation by the Nem1-Spo7 protein phosphatase complex. Pah1 is composed of a catalytic core (N-LIP and HAD-like domains, amphipathic helix, and the WRDPLVDID domain) and non-catalytic regulatory sequences (intrinsically disordered regions, RP domain, and acidic tail) for phosphorylation and interaction with Nem1-Spo7. In this work, the Pah1-CC (catalytic core) variant of Pah1 was constructed by molecular biological techniques that included PCR amplifications, ligase treatments, and plasmid construction. The fidelity of the construct was verified by DNA sequencing. Pah1-CC expressed on a low-copy plasmid complemented the pah1D mutant phenotypes of nuclear/ER membrane expansion, reduced levels of triacylglycerol, and lipid droplet formation without requiring the Nem1-Spo7 complex. The cellular function of Pah1-CC was supported by its PA phosphatase activity mostly associated with the membrane fraction. These findings on the Pah1 catalytic core enhance the understanding of its structural requirements for membrane localization and activity control. This research was supported by NIH grant GM136128.