Abstract
The Nem1-Spo7 protein phosphatase plays a role in lipid synthesis by controlling the membrane localization of Pah1, the diacylglycerol-producing phosphatidate (PA) phosphatase that is crucial for the synthesis of triacylglycerol in the yeast Saccharomyces cerevisiae By dephosphorylating Pah1, Nem1-Spo7 facilitates its translocation to the nuclear/endoplasmic reticulum membrane for catalytic activity. Like its substrate Pah1, Nem1-Spo7 is phosphorylated in the cell, but the specific protein kinases involved remain to be identified. In this study, we demonstrate that the Nem1-Spo7 complex is phosphorylated by protein kinase A (PKA), which is associated with active cell growth, metabolic activity, and membrane phospholipid synthesis. In vitro phosphorylation of purified Nem1-Spo7 and of their synthetic peptides revealed that both subunits of the phosphatase complex are PKA substrates. Using phosphoamino acid and phosphopeptide-mapping analyses coupled with site-directed mutagenesis, we identified Ser-140 and Ser-210 of Nem1 and Ser-28 of Spo7 as PKA-targeted phosphorylation sites. Immunodetection of the phosphatase complex from the cell with anti-PKA substrate antibody confirmed the in vivo phosphorylations of Nem1 and Spo7 on the serine residues. Lipid-labeling analysis of cells bearing phosphorylation-deficient alleles of NEM1 and SPO7 indicated that the PKA phosphorylation of the phosphatase complex stimulates phospholipid synthesis and attenuates the synthesis of triacylglycerol. This work advances the understanding of how PKA-mediated posttranslational modifications of Nem1 and Spo7 regulate lipid synthesis in yeast.
Highlights
The Nem1–Spo7 protein phosphatase plays a role in lipid synthesis by controlling the membrane localization of Pah1, the diacylglycerol-producing phosphatidate (PA) phosphatase that is crucial for the synthesis of triacylglycerol in the yeast Saccharomyces cerevisiae
We demonstrate that the Nem1–Spo7 complex is phosphorylated by protein kinase A (PKA), which is associated with active cell growth, metabolic activity, and membrane phospholipid synthesis
We demonstrated that the Nem1–Spo7 phosphatase complex is phosphorylated by PKA, a protein kinase whose activity is associated with active cell growth and increased metabolic activity and an increase in membrane phospholipid synthesis (10, 41, 42)
Summary
Molecular function is to catalyze the dephosphorylation of PA to form DAG (8, 9) (Fig. 1A). Along the lines of the latter phenotypes, mutants lacking Pah or the Nem1–Spo complex exhibit a defect in autophagy (35, 36) Taken together, these phenotypes emphasize the importance of the Nem1– Spo7/Pah phosphatase cascade in the regulation of lipid synthesis and cell physiology. The acidic pH optimum correlates with the intracellular pH (ϳ5) of yeast cells as they progress into the stationary phase (6, 37), the phase of growth when Pah function and TAG synthesis are maximal and the partitioning of PA toward lipid storage is favored over membrane phospholipid synthesis (24, 37, 38). Lipid compositional analyses of cells bearing phosphorylation-deficient mutations in Nem and Spo indicated that the PKA phosphorylation of the complex stimulates phospholipid synthesis and attenuates the synthesis of TAG. This work advances the understanding of the posttranslational modifications of Nem and Spo and the PKA-mediated regulation of lipid synthesis in yeast
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
