The CTR hydrophobic residues of Nem1 catalytic subunit are required to form a protein phosphatase complex with Spo7 to activate yeast Pah1 PA phosphatase.

Abstract

The Nem1-Spo7 phosphatase complex plays a key role in lipid metabolism as an activator of Pah1 phosphatidate phosphatase, which produces diacylglycerol for the synthesis of triacylglycerol and membrane phospholipids. For dephosphorylation of Pah1, the Nem1 catalytic subunit requires Spo7 for the recruitment of the protein substrate and interacts with the regulatory subunit through its conserved region (residues 251-446). In this work, we found that the Nem1 C-terminal region (CTR) (residues 414-436), which flanks the HAD-like catalytic domain (residues 251-413), contains the conserved hydrophobic residues (L414, L415, L417, L418, L421, V430, L434, and L436) that are necessary for the complex formation with Spo7. AlphaFold predicts that some CTR residues of Nem1 interact with Spo7 conserved regions, whereas some residues interact with the HAD-like domain. By site-directed mutagenesis, Nem1 variants were constructed to lack (Δ(414-446)) or substitute alanines (8A) and arginines (8R) for the hydrophobic residues. When coexpressed with Spo7, the CTR variants of Nem1 did not form a complex with Spo7. In addition, the Nem1 variants were incapable of catalyzing the dephosphorylation of Pah1 in the presence of Spo7. Moreover, the Nem1 variants expressed in nem1Δ cells did not complement the phenotypes characteristic of a defect in the Nem1-Spo7/Pah1 phosphatase cascade function (e.g., lipid synthesis, lipid droplet formation, and phospholipid biosynthetic gene expression). These findings support that Nem1 interacts with Spo7 through its CTR hydrophobic residues to form a phosphatase complex for catalytic activity and physiological functions.