Phosphatidate Phosphatase Activity Plays Key Role in Protection against Fatty Acid-induced Toxicity in Yeast

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 pah1⌬ 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 pah1⌬ 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 pah1⌬ 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 pah1⌬ mutant. However, the sensitivity of the pah1⌬ mutant to palmitoleic acid was not rescued by the dgk1⌬ mutation. Overall, these findings indicate a key role of PA phosphatase in TAG synthesis for protection against fatty acid-induced toxicity.PA 2 phosphatase (EC 3.1.3.4), which was first discovered by Kennedy and co-workers (1) in 1957, catalyzes the dephosphorylation of PA to produce DAG and P i (1) (Fig. 1). The reaction is dependent on Mg 2ϩ ions and is based on a DXDX(T/V) catalytic motif within a haloacid dehalogenase-like domain in the 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). PA, the enzyme substrate, is utilized for the synthesis of phospholipids via the liponucleotide intermediate CDP-DAG (7) (Fig. 1). Moreover, both PA (e.g. activation of cell growth, membrane proliferation, transcription, and vesicular trafficking) and DAG (e.g. activation of protein kinase C) have lipid signaling functions (8 -17), and PA phosphatase plays a role in controlling their cellular concentrations (2, 18). Thus, it is generally recognized that PA phosphatase is a key regulatory enzyme for controlling lipid metabolism and cell physiology (4, 7, 19 -21).The biochemistry and physiological roles of PA phosphatase emanated from studies in the model eukaryote yeast Saccharomyces cerevisiae and latterly in mammalian cells (4,7,19,21,22). PA phosphatase was first purified and characterized from yeast in 1989 (23), and the PAH1 4 gene encoding the enzyme was identified in 2006 (2). The discovery that PAH1 encodes PA phosphatase in yeast led to the revelation that the ...

Lipin-2 Reduces Proinflammatory Signaling Induced by Saturated Fatty Acids in Macrophages

“…Recently, it has been reported that deletion of yeast phosphatidate phosphatase makes the cells more sensitive to fatty acid-induced toxicity (38), a finding that concurs with the results presented here. Yeast possesses only one gene coding for phosphatidate phosphatase, whereas mammals possess three genes that encode for lipin proteins, and one of them, Lpin1, undergoes alternative splicing to generate two more lipin variants in mouse (lipin-1␣ and -1␤) and three in humans (lipin-1␣, -1␤, and -1␥).…”

Arabidopsis Lipins, PDAT1 Acyltransferase, and SDP1 Triacylglycerol Lipase Synergistically Direct Fatty Acids toward β-Oxidation, Thereby Maintaining Membrane Lipid Homeostasis

“…The increase in membrane lipid content was also observed in tgd1-1 disrupted in PDAT1 (Fan et al, 2013a) and in yeast mutants defective in TAG synthesis (Petschnigg et al, 2009;Fakas et al, 2011). Such an increase has been regarded as an alternative means to protect against lipotoxicity due to lipid overload (Petschnigg et al, 2009;Fakas et al, 2011).…”

“…The increase in membrane lipid content was also observed in tgd1-1 disrupted in PDAT1 (Fan et al, 2013a) and in yeast mutants defective in TAG synthesis (Petschnigg et al, 2009;Fakas et al, 2011). Such an increase has been regarded as an alternative means to protect against lipotoxicity due to lipid overload (Petschnigg et al, 2009;Fakas et al, 2011). Because phospholipid levels are elevated in both tgd1-1 sdp1-4, in which TAG hydrolysis is impaired, and tgd1-1 pxa1-2, in which FA degradation is impaired, it is unlikely that this increase is due to the augmented recycling to TAG into membrane lipids as a result of blocked FA turnover.…”

“…In yeast (Saccharomyces cerevisiae), a critical enzyme involved in TAG synthesis (Han et al, 2006) and LD formation (Adeyo et al, 2011) is the Mg 2+ -dependent phosphatidic acid phosphohydrolase (Pah1), the homolog of the mammalian lipin protein family. Yeast mutant cells lacking Pah1 display elevated phospholipid levels (Han et al, 2006), a massive proliferation of endoplasmic reticulum (ER) and nuclear membranes (Santos-Rosa et al, 2005), and increased sensitivity to exogenous FA-induced cell death (Fakas et al, 2011). Similarly, inactivation of the Caenorhabditis elegans lipin homolog has also been shown to cause decreased fat storage and altered ER membrane structure (Golden et al, 2009).…”

Phosphorylation of Dgk1 Diacylglycerol Kinase by Casein Kinase II Regulates Phosphatidic Acid Production in Saccharomyces cerevisiae

“…stationary phase) (6). Moreover, PA and DAG serve as signaling molecules that control transcription, membrane proliferation, vesicular trafficking, and the activation of cell growth (7-16).Disturbing the PA/DAG balance in yeast, as caused by the lack of Pah1 PA phosphatase activity, results in the abnormal regulation of phospholipid synthesis gene expression and phospholipid content, the aberrant growth of the nuclear/ER membrane, vacuole fragmentation, a defect in lipid droplet formation, an acute sensitivity to fatty acid-induced toxicity, and a reduction in chronological life span (1,3,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). Of the cellular defects imparted by the pah1⌬ mutation, those related to the elevated PA content are dependent on Dgk1 DAG kinase activity and are mimicked by the overexpression of DGK1 (1,3,6,18,21,23,25,27).…”