Abstract
The PAH1-encoded phosphatidate (PA) phosphatase in Saccharomyces cerevisiae is a pivotal enzyme that produces diacylglycerol for the synthesis of triacylglycerol (TAG) and simultaneously controls the level of PA used for phospholipid synthesis. Quantitative lipid analysis showed that the pah1Δ mutation caused a reduction in TAG mass and an elevation in the mass of phospholipids and free fatty acids, changes that were more pronounced in the stationary phase. The levels of unsaturated fatty acids in the pah1Δ mutant were unaltered, although the ratio of palmitoleic acid to oleic acid was increased with a similar change in the fatty acid composition of phospholipids. The pah1Δ mutant exhibited classic hallmarks of apoptosis in stationary phase and a marked reduction in the quantity of cytoplasmic lipid droplets. Cells lacking PA phosphatase were sensitive to exogenous fatty acids in the order of toxicity palmitoleic acid > oleic acid > palmitic acid. In contrast, the growth of wild type cells was not inhibited by fatty acid supplementation. In addition, wild type cells supplemented with palmitoleic acid exhibited an induction in PA phosphatase activity and an increase in TAG synthesis. Deletion of the DGK1-encoded diacylglycerol kinase, which counteracts PA phosphatase in controlling PA content, suppressed the defect in lipid droplet formation in the pah1Δ mutant. However, the sensitivity of the pah1Δ mutant to palmitoleic acid was not rescued by the dgk1Δ mutation. Overall, these findings indicate a key role of PA phosphatase in TAG synthesis for protection against fatty acid-induced toxicity.
Highlights
Enzyme (2– 4).3 The DAG produced by PA phosphatase is used for the synthesis of TAG and for the synthesis of PE and PC via the Kennedy pathway (4 –7) (Fig. 1)
The discovery that PAH1 encodes PA phosphatase in yeast led to the revelation that the lipodystrophic defect in the fatty liver dystrophy mouse (24, 25) was a PA phosphatase deficiency arising from mutations in the lpin1 gene (2, 26)
Since its discovery (1), researchers have asserted that PA phosphatase must play an important regulatory role in lipid metabolism because the enzyme is located at the branch point where PA is partitioned between the synthesis of TAG and the synthesis of membrane phospholipids (4, 7, 19 –21)
Summary
This process leads to the anchoring of dephosphorylated enzyme to the membrane via a short N-terminal amphipathic helix (33). Mechanisms that attenuate PA phosphatase must exist to regulate its functions in lipid metabolism These mechanisms include phosphorylation (33–35) and a limiting amount of Nem1p relative to Pah1p (34, 42) to control membrane association (33, 34). PA phosphatase acts at a pivotal nexus in lipid metabolism; depending on its activity, fatty acids are either channeled toward storage as TAG or to membrane assembly as phospholipids. This is exemplified by the phenotypes exhibited by yeast pah mutants that lack the enzyme. An analysis of lipid composition revealed that the pah1⌬ mutant exhibited a significant increase in the mass of membrane phospholipids and changes in their fatty acyl moieties when compared with wild type cells. PA phosphatase activity as opposed to a non-enzymatic function of Pah1p was essential in protecting cells from this toxicity
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