Protein Kinase A-mediated Phosphorylation of Pah1p Phosphatidate Phosphatase Functions in Conjunction with the Pho85p-Pho80p and Cdc28p-Cyclin B Kinases to Regulate Lipid Synthesis in Yeast

Pah1p, which functions as phosphatidate phosphatase (PAP) in the yeast Saccharomyces cerevisiae, plays a crucial role in lipid homeostasis by controlling the relative proportions of its substrate phosphatidate and its product diacylglycerol. The diacylglycerol produced by PAP is used for the synthesis of triacylglycerol as well as for the synthesis of phospholipids via the Kennedy pathway. Pah1p is a highly phosphorylated protein in vivo and has been previously shown to be phosphorylated by the protein kinases Pho85p-Pho80p and Cdc28p-cyclin B. In this work, we showed that Pah1p was a bona fide substrate for protein kinase A, and we identified by mass spectrometry and mutagenesis that Ser-10, Ser-677, Ser-773, Ser-774, and Ser-788 were the target sites of phosphorylation. Protein kinase A-mediated phosphorylation of Pah1p inhibited its PAP activity by decreasing catalytic efficiency, and the inhibitory effect was primarily conferred by phosphorylation at Ser-10. Analysis of the S10A and S10D mutations (mimicking dephosphorylation and phosphorylation, respectively), alone or in combination with the seven alanine (7A) mutations of the sites phosphorylated by Pho85p-Pho80p and Cdc28p-cyclin B, indicated that phosphorylation at Ser-10 stabilized Pah1p abundance and inhibited its association with membranes, PAP activity, and triacylglycerol synthesis. The S10A mutation enhanced the physiological effects imparted by the 7A mutations, whereas the S10D mutations attenuated the effects of the 7A mutations. These data indicated that the protein kinase A-mediated phosphorylation of Ser-10 functions in conjunction with the phosphorylations mediated by Pho85p-Pho80p and Cdc28p-cyclin B and that phospho-Ser-10 should be dephosphorylated for proper PAP function.     

Mutant phosphatidate phosphatase Pah1-W637A exhibits altered phosphorylation,membrane association,and enzyme function in yeast

The Saccharomyces cerevisiae PAH1-encoded phosphatidate (PA) phosphatase, which catalyzes the dephosphorylation of PA to produce diacylglycerol, controls the bifurcation of PA into triacylglycerol synthesis and phospholipid synthesis. Pah1 is inactive in the cytosol as a phosphorylated form and becomes active on the membrane as a dephosphorylated form by the Nem1–Spo7 protein phosphatase. We show that the conserved Trp-637 residue of Pah1, located in the intrinsically disordered region, is required for normal synthesis of membrane phospholipids, sterols, triacylglycerol, and the formation of lipid droplets. Analysis of mutant Pah1-W637A showed that the tryptophan residue is involved in the phosphorylation-mediated/dephosphorylation-mediated membrane association of the enzyme and its catalytic activity. The endogenous phosphorylation of Pah1-W637A was increased at the sites of the N-terminal region but was decreased at the sites of the C-terminal region. The altered phosphorylation correlated with an increase in its membrane association. In addition, membrane-associated PA phosphatase activity in vitro was elevated in cells expressing Pah1-W637A as a result of the increased membrane association of the mutant enzyme. However, the inherent catalytic function of Pah1 was not affected by the W637A mutation. Prediction of Pah1 structure by AlphaFold shows that Trp-637 and the catalytic residues Asp-398 and Asp-400 in the haloacid dehalogenase-like domain almost lie in the same plane, suggesting that these residues are important to properly position the enzyme for substrate recognition at the membrane surface. These findings underscore the importance of Trp-637 in Pah1 regulation by phosphorylation, membrane association of the enzyme, and its function in lipid synthesis.     

Phosphorylation Regulates the Ubiquitin-independent Degradation of Yeast Pah1 Phosphatidate Phosphatase by the 20S Proteasome

Saccharomyces cerevisiae Pah1 phosphatidate phosphatase, which catalyzes the conversion of phosphatidate to diacylglycerol for triacylglycerol synthesis and simultaneously controls phosphatidate levels for phospholipid synthesis, is subject to the proteasome-mediated degradation in the stationary phase of growth. In this study, we examined the mechanism for its degradation using purified Pah1 and isolated proteasomes. Pah1 expressed in S. cerevisiae or Escherichia coli was not degraded by the 26S proteasome, but by its catalytic 20S core particle, indicating that its degradation is ubiquitin-independent. The degradation of Pah1 by the 20S proteasome was dependent on time and proteasome concentration at the pH optimum of 7.0. The 20S proteasomal degradation was conserved for human lipin 1 phosphatidate phosphatase. The degradation analysis using Pah1 truncations and its fusion with GFP indicated that proteolysis initiates at the N- and C-terminal unfolded regions. The folded region of Pah1, in particular the haloacid dehalogenase-like domain containing the DIDGT catalytic sequence, was resistant to the proteasomal degradation. The structural change of Pah1, as reflected by electrophoretic mobility shift, occurs through its phosphorylation by Pho85-Pho80, and the phosphorylation sites are located within its N- and C-terminal unfolded regions. Phosphorylation of Pah1 by Pho85-Pho80 inhibited its degradation, extending its half-life by ∼2-fold. The dephosphorylation of endogenously phosphorylated Pah1 by the Nem1-Spo7 protein phosphatase, which is highly specific for the sites phosphorylated by Pho85-Pho80, stimulated the 20S proteasomal degradation and reduced its half-life by 2.6-fold. These results indicate that the proteolysis of Pah1 by the 20S proteasome is controlled by its phosphorylation state.     

Glycogen synthase kinase homolog Rim11 regulates lipid synthesis through the phosphorylation of Pah1 phosphatidate phosphatase in yeast

Pah1 phosphatidate (PA) phosphatase plays a major role in triacylglycerol synthesis in Saccharomyces cerevisiae by producing its precursor diacylglycerol and concurrently regulates de novo phospholipid synthesis by consuming its precursor PA. The function of Pah1 requires its membrane localization, which is controlled by its phosphorylation state. Pah1 is dephosphorylated by the Nem1-Spo7 protein phosphatase, whereas its phosphorylation occurs by multiple known and unknown protein kinases. In this work, we show that Rim11, a yeast homolog of mammalian glycogen synthase kinase-3β, is a protein kinase that phosphorylates Pah1 on serine (Ser12, Ser602, and Ser818) and threonine (Thr163, Thr164, Thr522) residues. Enzymological characterization of Rim11 showed that its K_m for Pah1 (0.4 μM) is similar to those of other Pah1-phosphorylating protein kinases, but its K_m for ATP (30 μM) is significantly higher than those of these same kinases. Furthermore, we demonstrate Rim11 phosphorylation of Pah1 does not require substrate prephosphorylation but was increased ∼2-fold upon its prephosphorylation by the Pho85-Pho80 protein kinase. In addition, we show Rim11-phosphorylated Pah1 was a substrate for dephosphorylation by Nem1-Spo7. Finally, we demonstrate the Rim11 phosphorylation of Pah1 exerted an inhibitory effect on its PA phosphatase activity by reduction of its catalytic efficiency. Mutational analysis of the major phosphorylation sites (Thr163, Thr164, and Ser602) indicated that Rim11-mediated phosphorylation at these sites was required to ensure Nem1-Spo7-dependent localization of the enzyme to the membrane. Overall, these findings advance our understanding of the phosphorylation-mediated regulation of Pah1 function in lipid synthesis.     

Regulation of lipid droplet and membrane biogenesis by the acidic tail of the phosphatidate phosphatase Pah1p

Lipins are evolutionarily conserved phosphatidate phosphatases that perform key functions in phospholipid, triglyceride, and membrane biogenesis. Translocation of lipins on membranes requires their dephosphorylation by the Nem1p-Spo7p transmembrane phosphatase complex through a poorly understood mechanism. Here we identify the carboxy-terminal acidic tail of the yeast lipin Pah1p as an important regulator of this step. Deletion or mutations of the tail disrupt binding of Pah1p to the Nem1p-Spo7p complex and Pah1p membrane translocation. Overexpression of Nem1p-Spo7p drives the recruitment of Pah1p in the vicinity of lipid droplets in an acidic tail–dependent manner and induces lipid droplet biogenesis. Genetic analysis shows that the acidic tail is essential for the Nem1p-Spo7p–dependent activation of Pah1p but not for the function of Pah1p itself once it is dephosphorylated. Loss of the tail disrupts nuclear structure, INO1 gene expression, and triglyceride synthesis. Similar acidic sequences are present in the carboxy-terminal ends of all yeast lipin orthologues. We propose that acidic tail–dependent binding and dephosphorylation of Pah1p by the Nem1p-Spo7p complex is an important determinant of its function in lipid and membrane biogenesis.     

Lipid partitioning at the nuclear envelope controls membrane biogenesis

Partitioning of lipid precursors between membranes and storage is crucial for cell growth, and its disruption underlies pathologies such as cancer, obesity, and type 2 diabetes. However, the mechanisms and signals that regulate this process are largely unknown. In yeast, lipid precursors are mainly used for phospholipid synthesis in nutrient-rich conditions in order to sustain rapid proliferation but are redirected to triacylglycerol (TAG) stored in lipid droplets during starvation. Here we investigate how cells reprogram lipid metabolism in the endoplasmic reticulum. We show that the conserved phosphatidate (PA) phosphatase Pah1, which generates diacylglycerol from PA, targets a nuclear membrane subdomain that is in contact with growing lipid droplets and mediates TAG synthesis. We find that cytosol acidification activates the master regulator of Pah1, the Nem1-Spo7 complex, thus linking Pah1 activity to cellular metabolic status. In the absence of TAG storage capacity, Pah1 still binds the nuclear membrane, but lipid precursors are redirected toward phospholipids, resulting in nuclear deformation and a proliferation of endoplasmic reticulum membrane. We propose that, in response to growth signals, activation of Pah1 at the nuclear envelope acts as a switch to control the balance between membrane biogenesis and lipid storage.     

Yeast Nem1-Spo7 Protein Phosphatase Activity on Pah1 Phosphatidate Phosphatase Is Specific for the Pho85-Pho80 Protein Kinase Phosphorylation Sites

Pah1 is the phosphatidate phosphatase in the yeast Saccharomyces cerevisiae that produces diacylglycerol for triacylglycerol synthesis and concurrently controls the levels of phosphatidate used for phospholipid synthesis. Phosphorylation and dephosphorylation of Pah1 regulate its subcellular location and phosphatidate phosphatase activity. Compared with its phosphorylation by multiple protein kinases, Pah1 is dephosphorylated by a protein phosphatase complex consisting of Nem1 (catalytic subunit) and Spo7 (regulatory subunit). In this work, we characterized the Nem1-Spo7 phosphatase complex for its enzymological, kinetic, and regulatory properties with phosphorylated Pah1. The dephosphorylation of Pah1 by Nem1-Spo7 phosphatase resulted in the stimulation (6-fold) of phosphatidate phosphatase activity. For Pah1 phosphorylated by the Pho85-Pho80 kinase complex, maximum Nem1-Spo7 phosphatase activity required Mg²⁺ ions (8 mm) and Triton X-100 (0.25 mm) at pH 5.0. The energy of activation for the reaction was 8.4 kcal/mol, and the enzyme was thermally labile at temperatures above 40 °C. The enzyme activity was inhibited by sodium vanadate, sodium fluoride, N-ethylmaleimide, and phenylglyoxal but was not significantly affected by lipids or nucleotides. Nem1-Spo7 phosphatase activity was dependent on the concentrations of Pah1 phosphorylated by Pho85-Pho80, Cdc28-cyclin B, PKA, and PKC with k_cat and K_m values of 0.29 s⁻¹ and 81 nm, 0.11 s⁻¹ and 127 nm, 0.10 s⁻¹ and 46 nm, and 0.02 s⁻¹ and 38 nm, respectively. Its specificity constant (k_cat/K_m) for Pah1 phosphorylated by Pho85-Pho80 was 1.6-, 4-, and 6-fold higher, respectively, than that phosphorylated by PKA, Cdc28-cyclin B, and PKC.     

An unconventional diacylglycerol kinase that regulates phospholipid synthesis and nuclear membrane growth

Changes in nuclear size and shape during the cell cycle or during development require coordinated nuclear membrane remodeling, but the underlying molecular events are largely unknown. We have shown previously that the activity of the conserved phosphatidate phosphatase Pah1p/Smp2p regulates nuclear structure in yeast by controlling phospholipid synthesis and membrane biogenesis at the nuclear envelope. Two screens for novel regulators of phosphatidate led to the identification of DGK1. We show that Dgk1p is a unique diacylglycerol kinase that uses CTP, instead of ATP, to generate phosphatidate. DGK1 counteracts the activity of PAH1 at the nuclear envelope by controlling phosphatidate levels. Overexpression of DGK1 causes the appearance of phosphatidate-enriched membranes around the nucleus and leads to its expansion, without proliferating the cortical endoplasmic reticulum membrane. Mutations that decrease phosphatidate levels decrease nuclear membrane growth in pah1Delta cells. We propose that phosphatidate metabolism is a critical factor determining nuclear structure by regulating nuclear membrane biogenesis.     

Roles of phosphatidate phosphatase enzymes in lipid metabolism

Phosphatidate phosphatase (PAP) enzymes catalyze the dephosphorylation of phosphatidate, yielding diacylglycerol and inorganic phosphate. In eukaryotic cells, PAP activity has a central role in the synthesis of phospholipids and triacylglycerol through its product diacylglycerol, and it also generates and/or degrades lipid-signaling molecules that are related to phosphatidate. There are two types of PAP enzyme, Mg(2+) dependent (PAP1) and Mg(2+) independent (PAP2), but only genes encoding PAP2 enzymes had been identified until recently, when a gene (PAH1) encoding a PAP1 enzyme was found in Saccharomyces cerevisiae. This discovery has revealed a molecular function of the mammalian protein lipin, a deficiency of which causes lipodystrophy in mice. With molecular information now available for both types of PAP, the specific roles of these enzymes in lipid metabolism are being clarified.     

Characterization of the S. cerevisiae inp51 mutant links phosphatidylinositol 4,5-bisphosphate levels with lipid content,membrane fluidity and cold growth

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] and its derivatives diphosphoinositol phosphates (DPIPs) play key signaling and regulatory roles. However, a direct function of these molecules in lipid and membrane homeostasis remains obscure. Here, we have studied the cold tolerance phenotype of yeast cells lacking the Inp51-mediated phosphoinositide-5-phosphatase. Genetic and biochemical approaches showed that increased metabolism of PI(4,5)P₂ reduces the activity of the Pho85 kinase by increasing the levels of the DPIP isomer 1-IP₇. This effect was key in the cold tolerance phenotype. Indeed, pho85 mutant cells grew better than the wild-type at 15 °C, and lack of this kinase abolished the inp51-mediated cold phenotype. Remarkably, reduced Pho85 function by loss of Inp51 affected the activity of the Pho85-regulated target Pah1, the yeast phosphatidate phosphatase. Cells lacking Inp51 showed reduced Pah1 abundance, derepression of an INO1-lacZ reporter, decreased content of triacylglycerides and elevated levels of phosphatidate, hallmarks of the pah1 mutant. However, the inp51 phenotype was not associated to low Pah1 activity since deletion of PAH1 caused cold sensitivity. In addition, the inp51 mutant exhibited features not shared by pah1, including a 40%-reduction in total lipid content and decreased membrane fluidity. These changes may influence the activity of membrane-anchored and/or associated proteins since deletion of INP51 slows down the transit to the vacuole of the fluorescent dye FM4-64. In conclusion, our work supports a model in which changes in the PI(4,5)P₂ pool affect the 1-IP₇ levels modulating the activity of Pho85, Pah1 and likely additional Pho85-controlled targets, and regulate lipid composition and membrane properties.     

A review of phosphatidate phosphatase assays

Phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol and regulates the synthesis of membrane phospholipids. There is much interest in this enzyme because it controls the cellular levels of its substrate, phosphatidate (PA), and product, DAG; defects in the metabolism of these lipid intermediates are the basis for lipid-based diseases such as obesity, lipodystrophy, and inflammation. The measurement of PAP activity is required for studies aimed at understanding its mechanisms of action, how it is regulated, and for screening its activators and/or inhibitors. Enzyme activity is determined through the use of radioactive and nonradioactive assays that measure the product, DAG, or P_i. However, sensitivity and ease of use are variable across these methods. This review summarizes approaches to synthesize radioactive PA, to analyze radioactive and nonradioactive products, DAG and P_i, and discusses the advantages and disadvantages of each PAP assay.     

Yeast lipin 1 orthologue pah1p regulates vacuole homeostasis and membrane fusion

Vacuole homotypic fusion requires a group of regulatory lipids that includes diacylglycerol, a fusogenic lipid that is produced through multiple metabolic pathways including the dephosphorylation of phosphatidic acid (PA). Here we examined the relationship between membrane fusion and PA phosphatase activity. Pah1p is the single yeast homologue of the Lipin family of PA phosphatases. Deletion of PAH1 was sufficient to cause marked vacuole fragmentation and abolish vacuole fusion. The function of Pah1p solely depended on its phosphatase activity as complementation studies showed that wild type Pah1p restored fusion, whereas the phosphatase dead mutant Pah1p(D398E) had no effect. We discovered that the lack of PA phosphatase activity blocked fusion by inhibiting the binding of SNAREs to Sec18p, an N-ethylmaleimide-sensitive factor homologue responsible for priming inactive cis-SNARE complexes. In addition, pah1Δ vacuoles were devoid of the late endosome/vacuolar Rab Ypt7p, the phosphatidylinositol 3-kinase Vps34p, and Vps39p, a subunit of the HOPS (homotypic fusion and vacuole protein sorting) tethering complex, all of which are required for vacuole fusion. The lack of Vps34p resulted in the absence of phosphatidylinositol 3-phosphate, a lipid required for SNARE activity and vacuole fusion. These findings demonstrate that Pah1p and PA phosphatase activity are critical for vacuole homeostasis and fusion.     

The cellular functions of the yeast lipin homolog PAH1p are dependent on its phosphatidate phosphatase activity

The Saccharomyces cerevisiae PAH1-encoded Mg2+-dependent phosphatidate phosphatase (PAP1, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol and Pi. This enzyme plays a major role in the synthesis of triacylglycerols and phospholipids in S. cerevisiae. PAP1 contains the DXDX(T/V) catalytic motif (DIDGT at residues 398-402) that is shared by the mammalian fat-regulating protein lipin 1 and the superfamily of haloacid dehalogenase-like proteins. The yeast enzyme also contains a conserved glycine residue (Gly80) that is essential for the fat-regulating function of lipin 1 in a mouse model. In this study, we examined the roles of the putative catalytic motif and the conserved glycine for PAP1 activity by a mutational analysis. The PAP1 activities of the D398E and D400E mutant enzymes were reduced by >99.9%, and the activity of the G80R mutant enzyme was reduced by 98%. The mutant PAH1 alleles whose products lacked PAP1 activity were nonfunctional in vivo and failed to complement the pah1Delta mutant phenotypes of temperature sensitivity, respiratory deficiency, nuclear/endoplasmic reticulum membrane expansion, derepression of INO1 expression, and alterations in lipid composition. These results demonstrated that the PAP1 activity of the PAH1 gene product is essential for its roles in lipid metabolism and cell physiology.     

TORC1 Regulates Pah1 Phosphatidate Phosphatase Activity via the Nem1/Spo7 Protein Phosphatase Complex

The evolutionarily conserved target of rapamycin complex 1 (TORC1) controls growth-related processes such as protein, nucleotide, and lipid metabolism in response to growth hormones, energy/ATP levels, and amino acids. Its deregulation is associated with cancer, type 2 diabetes, and obesity. Among other substrates, mammalian TORC1 directly phosphorylates and inhibits the phosphatidate phosphatase lipin-1, a central enzyme in lipid metabolism that provides diacylglycerol for the synthesis of membrane phospholipids and/or triacylglycerol as neutral lipid reserve. Here, we show that yeast TORC1 inhibits the function of the respective lipin, Pah1, to prevent the accumulation of triacylglycerol. Surprisingly, TORC1 regulates Pah1 in part indirectly by controlling the phosphorylation status of Nem1 within the Pah1-activating, heterodimeric Nem1-Spo7 protein phosphatase module. Our results delineate a hitherto unknown TORC1 effector branch that controls lipin function in yeast, which, given the recent discovery of Nem1-Spo7 orthologous proteins in humans, may be conserved.