The Nem1-Spo7 protein phosphatase complex plays a pivotal role in the lipid metabolism of Saccharomyces cerevisiae by activating Pah1, which catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol for the synthesis of triacylglycerol and the phospholipids phosphatidylcholine and phosphatidylethanolamine. As the catalytic subunit, Nem1 dephosphorylates Pah1 to facilitate its membrane localization for phosphatidate phosphatase activity. In this study, we investigated the functional importance of the Nem1 residues (Asp-257, Asp-259, Thr-261, Thr-319, Lys-360, Asp-386, and Asn-387) conserved in the four active site motifs of the haloacid dehalogenase (HAD)-like domain. The effects of alanine mutations of the active site residues on Pah1 dephosphorylation and associated functions in lipid synthesis were examined. Our results showed that the Nem1 active site mutants are catalytically inactive without a defect in protein stability, complex formation with Spo7, and recruitment of Pah1 to the membrane. Cells expressing the Nem1 active site mutants are defective in the activation of Pah1 for triacylglycerol synthesis and the regulation of phosphatidylserine synthase Cho1 expression. These findings demonstrate that the active site motifs of Nem1 are important for catalytic activity, which is required for the full activation of Pah1 and its role in cellular lipid homeostasis.

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Journal Article

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Bostrom ER, Jog R, Stukey GJ, Han GS, Carman GM

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