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 haloacid dehalogenase–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 haloacid dehalogenase–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 co-expressed 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.

中文翻译：

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Nem1 催化亚基的 CTR 疏水残基需要与 Spo7 形成蛋白质磷酸酶复合物，以激活酵母 Pah1 PA 磷酸酶


Nem1-Spo7 磷酸酶复合物作为 Pah1 磷酸盐磷酸酶的激活剂在脂质代谢中起关键作用，Pah1 磷酸盐磷酸酶产生甘油二酰基甘油，用于合成三酰甘油和膜磷脂。对于 Pah1 的去磷酸化，Nem1 催化亚基需要 Spo7 来募集蛋白质底物，并通过其保守区域（残基 251-446）与调节亚基相互作用。在这项工作中，我们发现位于卤酸脱卤酶样催化结构域（残基 251-413）两侧的 Nem1 C 末端区域 （CTR）（残基 414-436）包含保守的疏水残基（L414、L415、L417、L418、L421、V430、L434 和 L436）是与 Spo7 形成复合物所必需的。AlphaFold 预测 Nem1 的一些 CTR 残基与 Spo7 保守区域相互作用，而一些残基与卤酸脱卤酶样结构域相互作用。通过定点诱变，Nem1 变体被构建为缺乏 （Δ（414–446）） 或替代丙氨酸 （8A） 和精氨酸 （8R） 作为疏水残基。当与 Spo7 共表达时，Nem1 的 CTR 变体不会与 Spo7 形成复合物。此外，在 Spo7 存在下，Nem1 变体无法催化 Pah1 的去磷酸化。此外，在 nem1Δ 细胞中表达的 Nem1 变体并不补充 Nem1-Spo7/Pah1 磷酸酶级联功能缺陷的表型特征（例如，脂质合成、脂滴形成和磷脂生物合成基因表达）。这些发现支持 Nem1 通过其 CTR 疏水残基与 Spo7 相互作用，形成磷酸酶复合物，用于催化活性和生理功能。