Abstract
The Saccharomyces cerevisiae PAH1-encoded phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol and plays a role in the transcriptional regulation of phospholipid synthesis genes. PAP is phosphorylated at multiple Ser and Thr residues and is dephosphorylated for in vivo function by the Nem1p-Spo7p protein phosphatase complex localized in the nuclear/endoplasmic reticulum membrane. In this work, we characterized seven previously identified phosphorylation sites of PAP that are within the Ser/Thr-Pro motif. When expressed on a low copy plasmid, wild type PAP could not complement the pah1Δ mutant in the absence of the Nem1p-Spo7p complex. However, phosphorylation-deficient PAP (PAP-7A) containing alanine substitutions for the seven phosphorylation sites bypassed the requirement of the phosphatase complex and complemented the pah1Δ nem1Δ mutant phenotypes, such as temperature sensitivity, nuclear/endoplasmic reticulum membrane expansion, decreased triacylglycerol synthesis, and derepression of INO1 expression. Subcellular fractionation coupled with immunoblot analysis showed that PAP-7A was highly enriched in the membrane fraction. In fluorescence spectroscopy analysis, the PAP-7A showed tighter association with phospholipid vesicles than wild type PAP. Using site-directed mutagenesis of PAP, we identified Ser(602), Thr(723), and Ser(744), which belong to the seven phosphorylation sites, as the sites phosphorylated by the CDC28 (CDK1)-encoded cyclin-dependent kinase. Compared with the dephosphorylation mimic of the seven phosphorylation sites, alanine substitution for Ser(602), Thr(723), and/or Ser(744) had a partial effect on circumventing the requirement for the Nem1p-Spo7p complex.
Highlights
The Saccharomyces cerevisiae PAH1-encoded phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol and plays a role in the transcriptional regulation of phospholipid synthesis genes
Using site-directed mutagenesis of PAP, we identified Ser602, Thr723, and Ser744, which belong to the seven phosphorylation sites, as the sites phosphorylated by the CDC28 (CDK1)-encoded cyclin-dependent kinase
Phosphorylation of PAP by CDK—Data indicate that CDC28 (CDK1)-encoded CDK might be the protein kinase responsible for the phosphorylation of the seven sites within the Ser/ Thr-Pro motif of PAP previously shown to be phosphorylated in vivo [5, 25]
Summary
The Saccharomyces cerevisiae PAH1-encoded phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol and plays a role in the transcriptional regulation of phospholipid synthesis genes. Phosphorylation-deficient PAP (PAP-7A) containing alanine substitutions for the seven phosphorylation sites bypassed the requirement of the phosphatase complex and complemented the pah1⌬ nem1⌬ mutant phenotypes, such as temperature sensitivity, nuclear/endoplasmic reticulum membrane expansion, decreased triacylglycerol synthesis, and derepression of INO1 expression. In the yeast Saccharomyces cerevisiae, the PAH1-encoded phosphatidate phosphatase (PAP) catalyzes the dephosphorylation of PA, yielding DAG and Pi [1, 2] This reaction is dependent on Mg2ϩ ions and is based on a DXDX(T/V) catalytic motif within a haloacid dehalogenase-like domain in the enzyme [2,3,4]. The nine remaining sites are putative targets for protein kinases, such as protein kinases A and C, casein kinases I and II, and MAPK, indicating that the regulation of PAP by phosphorylation is complex
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