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![Effects of prephosphorylation of Pah1 by CKII, Pho85-Pho80, PKA, and PKC on subsequent phosphorylation by CKI. Unphosphorylated Pah1 (10 g/ml) was prephosphorylated by the indicated amounts of CKII (A), Pho85-Pho80 (B), PKA (C), or PKC (D) for 2 h with 100 M ATP. The prephosphorylated Pah1 was then phosphorylated by 6 fmol/min of CKI with [-32 P]ATP for 2 h. The 32 P-labeled Pah1 was separated from labeled ATP by SDS-PAGE and subjected to phosphorimaging and ImageQuant analysis. The amount of the phosphorylated Pah1 that was not subjected to prephosphorylation by CKII, Pho85-Pho80, PKA, or PKC was set at 100%. The data reported are the result of two independent experiments and the line drawn represents the average of the two experiments. 7A (black circle), Pho85-Pho80 phosphorylation site mutations.](profile/Lu-Sheng-Hsieh/publication/336764075/figure/fig3/AS:817357932986384@1571884725760/Effects-of-prephosphorylation-of-Pah1-by-CKII-Pho85-Pho80-PKA-and-PKC-on-subsequent.png)
Effects of prephosphorylation of Pah1 by CKII, Pho85-Pho80, PKA, and PKC on subsequent phosphorylation by CKI. Unphosphorylated Pah1 (10 g/ml) was prephosphorylated by the indicated amounts of CKII (A), Pho85-Pho80 (B), PKA (C), or PKC (D) for 2 h with 100 M ATP. The prephosphorylated Pah1 was then phosphorylated by 6 fmol/min of CKI with [-32 P]ATP for 2 h. The 32 P-labeled Pah1 was separated from labeled ATP by SDS-PAGE and subjected to phosphorimaging and ImageQuant analysis. The amount of the phosphorylated Pah1 that was not subjected to prephosphorylation by CKII, Pho85-Pho80, PKA, or PKC was set at 100%. The data reported are the result of two independent experiments and the line drawn represents the average of the two experiments. 7A (black circle), Pho85-Pho80 phosphorylation site mutations.
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The PAH1-encoded phosphatidate phosphatase in Saccharomyces cerevisiae plays a major role in triacylglycerol synthesis and the control of phospholipid synthesis. For its catalytic function on the nuclear/endoplasmic reticulum membrane, Pah1 translocates to the membrane through its phosphorylation/dephosphorylation. Pah1 phosphorylation on multiple...
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... reciprocal experiments, we examined whether the prephosphorylation of Pah1 by CKII, Pho85-Pho80, PKA, and PKC affects the subsequent phosphorylation by CKI. The prephosphorylation of Pah1 by Pho85-Pho80 caused a dose-dependent inhibition (75%) of its phosphorylation by CKI (Fig. 7B). However, the prephosphorylation of Pah1 by the other protein kinases had no or little effect on the subsequent phosphorylation of Pah1 by CKI (Fig. 7A, C, and D). As discussed above, CKI and Pho85-Pho80 share common target sites (Fig. 1B), and thus the inhibitory effect of Pho85-Pho80 on the CKI activity was considered to be caused by ...
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... CKII, Pho85-Pho80, PKA, and PKC affects the subsequent phosphorylation by CKI. The prephosphorylation of Pah1 by Pho85-Pho80 caused a dose-dependent inhibition (75%) of its phosphorylation by CKI (Fig. 7B). However, the prephosphorylation of Pah1 by the other protein kinases had no or little effect on the subsequent phosphorylation of Pah1 by CKI (Fig. 7A, C, and D). As discussed above, CKI and Pho85-Pho80 share common target sites (Fig. 1B), and thus the inhibitory effect of Pho85-Pho80 on the CKI activity was considered to be caused by a reduction in available target residues. To test this possibility, we analyzed the CKI-phosphorylation of Pah1-7A, which has alanine substitutions for ...
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... on the CKI activity was considered to be caused by a reduction in available target residues. To test this possibility, we analyzed the CKI-phosphorylation of Pah1-7A, which has alanine substitutions for the seven target sites of Pho85-Pho80 (40). The 7A mutations reduced (40%) the inhibitory effect of the prephosphorylation by Pho85-Pho80 (Fig. 7B). Thus, the inhibition of the CKI-mediated phosphorylation is due to the phosphorylation by Pho85-Pho80 of sites common to both protein kinases. However, the data also indicate that phosphorylation by Pho85-Pho80 at sites that are not common to CKI affect the phosphorylation by ...
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... reciprocal experiments, we examined whether the prephosphorylation of Pah1 by CKII, Pho85-Pho80, PKA, and PKC affects the subsequent phosphorylation by CKI. The prephosphorylation of Pah1 by Pho85-Pho80 caused a dose-dependent inhibition (75%) of its phosphorylation by CKI (Fig. 7B). However, the prephosphorylation of Pah1 by the other protein kinases had no or little effect on the subsequent phosphorylation of Pah1 by CKI (Fig. 7A, C, and D). As discussed above, CKI and Pho85-Pho80 share common target sites (Fig. 1B), and thus the inhibitory effect of Pho85-Pho80 on the CKI activity was considered to be caused by ...
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... CKII, Pho85-Pho80, PKA, and PKC affects the subsequent phosphorylation by CKI. The prephosphorylation of Pah1 by Pho85-Pho80 caused a dose-dependent inhibition (75%) of its phosphorylation by CKI (Fig. 7B). However, the prephosphorylation of Pah1 by the other protein kinases had no or little effect on the subsequent phosphorylation of Pah1 by CKI (Fig. 7A, C, and D). As discussed above, CKI and Pho85-Pho80 share common target sites (Fig. 1B), and thus the inhibitory effect of Pho85-Pho80 on the CKI activity was considered to be caused by a reduction in available target residues. To test this possibility, we analyzed the CKI-phosphorylation of Pah1-7A, which has alanine substitutions for ...
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... on the CKI activity was considered to be caused by a reduction in available target residues. To test this possibility, we analyzed the CKI-phosphorylation of Pah1-7A, which has alanine substitutions for the seven target sites of Pho85-Pho80 (40). The 7A mutations reduced (40%) the inhibitory effect of the prephosphorylation by Pho85-Pho80 (Fig. 7B). Thus, the inhibition of the CKI-mediated phosphorylation is due to the phosphorylation by Pho85-Pho80 of sites common to both protein kinases. However, the data also indicate that phosphorylation by Pho85-Pho80 at sites that are not common to CKI affect the phosphorylation by ...
Citations
... Cells that lack either paralog are viable, but loss of both is lethal. Yck1/2 are thought to influence diverse cellular processes, such as cell growth, endocytosis, vesicle trafficking, cell cycle progression and glucose sensing (Gadura et al., 2006;Hassaninasab et al., 2019;Moriya and Johnston, 2004;Panek, 1997;Pasula et al., 2010;Robinson et al., 1993;Snowdon and Johnston, 2016;Stalder et al., 2016). ...
Tor kinases play diverse and essential roles in control of nutrient signaling and cell growth. Tor kinases are assembled into two large multiprotein complexes referred to as Tor Complex 1 and Tor Complex 2 (TORC1 and TORC2). In budding yeast, TORC2 controls a signaling network that relays signals regarding carbon source that strongly influence growth rate and cell size. However, the mechanisms that control TORC2 signaling are poorly understood. Activation of TORC2 requires Mss4, a phosphoinositol kinase that initiates assembly of a multi protein complex at the plasma membrane that recruits and activates downstream targets of TORC2. Localization of Mss4 to the plasma membrane is controlled by phosphorylation and previous work suggested that yeast homologs of casein kinase 1g, referred to as Yck1 and Yck2, control phosphorylation of Mss4. Here, we generated a new analog-sensitive allele of YCK2 and used it to test whether Yck1/2 influence signaling in the TORC2 network. We found that multiple components of the TORC2 network are strongly influenced by Yck1/2 signaling.
... Pah1 expression is regulated by growth phase and nutrient status; increased expression as mediated by nutrient depletion in stationary phase is coincident with PA utilization for TAG synthesis, whereas reduced expression as mediated by nutrient sufficiency in exponential phase is coincident with PA utilization for phospholipid synthesis (28,56). The PAP activity of Pah1 is modulated by lipids (57,58), nucleotides (59), and the phosphorylation status (60)(61)(62)(63)(64)(65)(66)(67)(68)(69). For example, PAP activity is stimulated by CDP-DAG and PI (57), whereas the enzyme activity is inhibited by sphingoid bases (58) and the nucleotides ATP and CTP (59). ...
... For example, PAP activity is stimulated by CDP-DAG and PI (57), whereas the enzyme activity is inhibited by sphingoid bases (58) and the nucleotides ATP and CTP (59). Multiple protein kinases phosphorylate Pah1 (62)(63)(64)(65)(66)(67)(68), whereas the phosphorylated enzyme is dephosphorylated by the Nem1 (catalytic subunit)-Spo7 (regulatory subunit) phosphatase complex (60,69,70). Whereas some of its phosphorylations (e.g., by Pho85-Pho80 and Rim11) inhibit PAP activity (63,68), the dephosphorylation by Nem1-Spo7 stimulates activity (63,69). ...
... In the second assay, the enzyme activity was measured by following the release of 32 P i from 32 P-labeled Pah1 (69). As a substrate of Nem1-Spo7, two forms of phosphorylated Pah1 were used in this study: (1) Pah1 prepared from yeast (61,(98)(99)(100)(101)(102)(103)(104)(105)(106)(107)(108)(109)(110), which is endogenously phosphorylated by multiple protein kinases (62)(63)(64)(65)(66)(67); (2) Pah1 heterologously expressed in E. coli and phosphorylated in vitro by the Pho85-Pho80 protein kinase (63). ...
In the yeast Saccharomyces cerevisiae, the PAH1-encoded Mg²⁺-dependent phosphatidate (PA) phosphatase (a.k.a. Pah1) regulates the bifurcation of PA to diacylglycerol for triacylglycerol synthesis and to CDP-diacylglycerol for phospholipid synthesis. Pah1 function is mainly regulated via control of its cellular location by phosphorylation and dephosphorylation. Pah1 phosphorylated by multiple protein kinases is sequestered in the cytosol apart from its substrate PA in the membrane. The phosphorylated Pah1 is then recruited and dephosphorylated by the protein phosphatase complex Nem1 (catalytic subunit)-Spo7 (regulatory subunit) in the endoplasmic reticulum membrane. The dephosphorylated Pah1 hops onto and scoots along the membrane to recognize PA for its dephosphorylation to diacylglycerol. Here, we developed a proteoliposome model system that mimics the Nem1-Spo7/Pah1 phosphatase cascade to provide a tool for studying Pah1 regulation. Purified Nem1-Spo7 was reconstituted into phospholipid vesicles prepared in accordance with the phospholipid composition of the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7 phosphatase reconstituted in the proteoliposomes, which were measured 60 nm in an average diameter, was catalytically active on Pah1 phosphorylated by Pho85-Pho80, and its active site was located at the external side of the phospholipid bilayer. Moreover, we determined that PA stimulated the Nem1-Spo7 activity, and the regulatory effect was governed by the nature of the phosphate headgroup but not by the fatty acyl moiety of PA. The reconstitution system for the Nem1-Spo7/Pah1 phosphatase cascade, which starts with the phosphorylation of Pah1 by Pho85-Pho80 and ends with the production of diacylglycerol, is a significant advance to understand a regulatory cascade in yeast lipid synthesis.
... The cellular function of Pah1 is primarily regulated by its localization. Following its expression that is controlled by nutrients at the transcriptional level (11,32), Pah1 is phosphorylated on the serine and threonine residues (33-46) by many protein kinases (47)(48)(49)(50)(51)(52). The phosphorylated form of Pah1 is stable in the cytosol but nonfunctional due to the lack of its association with the PA-containing membrane. ...
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. Whereas Pah1 is dephosphorylated by the Nem1-Spo7 protein phosphatase, 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 (Ser-12, Ser-602, and Ser-818) and threonine (Thr-163, Thr-164, Thr-522) residues. Enzymological characterization of Rim11 showed that its Km for Pah1 (0.4 μM) is similar to those of other Pah1-phosphorylating protein kinases, but its Km 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 (Thr-163, Thr-164, and Ser-602) 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.
... As depicted in Fig. 1, the function of Pah1 as a lipid biosynthetic enzyme is mainly controlled by its localization. Following its expression, which is regulated at the level of transcription by nutrient status Soto-Cardalda et al., 2011), Pah1 in the cytosol is phosphorylated on serine and threonine residues (Albuquerque et al., 2008;Bodenmiller et al., 2010;Chi et al., 2007;Gnad et al., 2009;Gruhler et al., 2005;Helbig et al., 2010;Lanz et al., 2021;Li et al., 2007;MacGilvray et al., 2020;O'Hara et al., 2006;Smolka et al., 2007;Soufi et al., 2009;Soulard et al., 2010;Swaney et al., 2013) by multiple protein kinases (Choi et al., , 2012Hassaninasab et al., 2019;Hsieh et al., 2016;Su et al., 2012Su et al., , 2014a. Phosphorylated Pah1 is non-functional because it is sequestered in the cytosol apart from its membrane-associated substrate PA. ...
... A peptide containing a specific phosphorylation site would be expected to serve as a substrate for the protein kinase that phosphorylates that site. In fact, Pah1 peptides that contain a specific phosphorylation site for CKI (Ser-511, residues 506-LYFEDSDNEVDT-517) and PKC (Ser-769, residues 763-NYNRTKSRRA-772) are substrates for CKI and PKC, respectively (Dey et al., 2017;Hassaninasab et al., 2019). Likewise, Nem1 peptides with specific phosphorylation sites for PKA (Ser-140, residues 135-KRNRGSNASEN-145 and Ser-210, residues 205-RPRSYSKSELS-215) and PKC are substrates for PKA and PKC, respectively (Dey et al., 2019;Su et al., 2018). ...
... Likewise, Nem1 peptides with specific phosphorylation sites for PKA (Ser-140, residues 135-KRNRGSNASEN-145 and Ser-210, residues 205-RPRSYSKSELS-215) and PKC are substrates for PKA and PKC, respectively (Dey et al., 2019;Su et al., 2018). Substitution of a non-phosphorylatable alanine residue for the phosphorylatable serine residues (shown in bold red color) in these peptide substrates obviates the phosphorylation by the protein kinase involved (Dey et al., 2019;Hassaninasab et al., 2019;Su et al., 2018). ...
The PAH1-encoded phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol, controls the divergence of phosphatidate into triacylglycerol synthesis and phospholipid synthesis. Pah1 is inactive in the cytosol as a phosphorylated form and becomes active on the nuclear/endoplasmic reticulum membrane as a dephosphorylated form by the Nem1-Spo7 protein phosphatase complex. The phosphorylation of Pah1 by protein kinases, which include casein kinases I and II, Pho85-Pho80, Cdc28-cyclin B, and protein kinases A and B, controls its cellular location, catalytic activity, and susceptibility to proteasomal degradation. Nem1 (catalytic subunit) and Spo7 (regulatory subunit), which form a protein phosphatase complex catalyzing the dephosphorylation of Pah1 for its activation, are phosphorylated by protein kinases A and C. In this review, we discuss the functions and interrelationships of the protein kinases in the control of the Nem1-Spo7/Pah1 phosphatase cascade and lipid synthesis.
... The PAP activity of Pah1 is stimulated by negatively charged phospholipids (15), but inhibited by positively charged sphingoid bases (16) and nucleotides (17). Phosphorylation is a major posttranslational modification for Pah1, and it is highly phosphorylated (18)(19)(20)(21)(22)(23)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32) by multiple protein kinases (33)(34)(35)(36)(37)(38) (Fig. 2). Some of its phosphorylations are hierarchical in nature (e.g., phosphorylation on one site affects phosphorylation on another site), whereas other phosphorylations occur on common sites by different protein kinases (7,12). ...
... Most Pah1-interacting proteins identified by physical interaction studies are the plethora of protein kinases (33)(34)(35)(36)(37)(38)(93)(94)(95)(96) (Fig. 10). According to this model, Trp-637 and Trp-873, and their respective catalytic residues (i.e., Asp-398 and Asp-400 in yeast Pah1 and Asp-678 and Asp-680 in human lipin 1) almost lie in the same plane, suggesting that the tryptophan residues are important to properly position the catalytic residues for substrate recognition at the membrane surface. ...
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 complex. 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/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/dephosphorylation regulates the subcellular localization, protein abundance, and PAP activity of Pah1 [19,20]. The newly synthesized Pah1 protein is localized in the cytosol, and is phosphorylated by multiple protein kinases [21][22][23] such as CDC28-cyclin B [24], Pho85-Pho80 [19], protein kinase A [25], protein kinase C [26], casein kinase II [27], and casein kinase I [28]. Phosphorylated Pah1 is recruited to the endoplasmic reticulum (ER) membrane to be dephosphorylated by the Nem1-Spo7 protein phosphatase [21,29,30]. ...
Saccharomyces cerevisiae Pah1 phosphatidate phosphatase (PAP) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol, controlling phospholipids and triacylglycerol metabolisms. Pah1 and human Lipin 1 are intrinsically disordered proteins with 56% and 43% unfolded regions, respectively. Truncation analysis of the conserved and non-conserved regions showed that N- and C-conserved regions are essential for the catalytic activity of Pah1. PAP activities can be detected in the conserved N-terminal Lipin (NLIP) domain and C-terminal Lipin (CLIP)/haloacid dehalogenase (HAD)-like domain of Pah1 and Lipin 1, suggesting that the evolutionarily conserved domains are essential for the catalytic activity. The removal of disordered hydrophilic regions drastically reduced the protein solubility of Pah1. Thioredoxin is an efficient fusion protein for production of soluble NLIP–HAD recombinant proteins in Escherichia coli.
... The PAP enzymes are divided into Mg 2+ -dependent PAP1 or Mg 2+ -independent PAP2 [also be called lipid phosphate phosphatase (LPP) or diacylglycerol pyrophosphate (DGPP) phosphatase] based on the cofactor requirement for catalytic activity (Jamal et al., 1991;Brindley et al., 2002). The PAP1 enzymes play roles in cell homeostasis and lipid synthesis (Han et al., 2006;Sherr et al., 2017;Hassaninasab et al., 2019), and PAP1 enzyme, PAH1, performs catalytic function to regulate phospholipid synthesis on the nuclear and endoplasmic reticulum (Eastmond et al., 2010;Hassaninasab et al., 2019). The absence of Pahp1 (encoded PAH1) leads to the upregulated of V-ATPase (Sherr et al., 2017). ...
... The PAP enzymes are divided into Mg 2+ -dependent PAP1 or Mg 2+ -independent PAP2 [also be called lipid phosphate phosphatase (LPP) or diacylglycerol pyrophosphate (DGPP) phosphatase] based on the cofactor requirement for catalytic activity (Jamal et al., 1991;Brindley et al., 2002). The PAP1 enzymes play roles in cell homeostasis and lipid synthesis (Han et al., 2006;Sherr et al., 2017;Hassaninasab et al., 2019), and PAP1 enzyme, PAH1, performs catalytic function to regulate phospholipid synthesis on the nuclear and endoplasmic reticulum (Eastmond et al., 2010;Hassaninasab et al., 2019). The absence of Pahp1 (encoded PAH1) leads to the upregulated of V-ATPase (Sherr et al., 2017). ...
The gene encoding a putative phosphatidate phosphatase (PAP) from tolerant saline-alkali (TSA) Chlorella, ChPAP, was identified from a yeast cDNA library constructed from TSA Chlorella after a NaCl treatment. ChPAP expressed in yeast enhanced its tolerance to NaCl and sorbitol. The ChPAP protein from a GFP-tagged construct localized to the plasma membrane and the lumen of vacuoles. The relative transcript levels of ChPAP in Chlorella cells were strongly induced by NaCl and sorbitol as assessed by northern blot analyses. Thus, ChPAP may play important roles in promoting Na-ion movement into the cell and maintaining the cytoplasmic ion balance. In addition, ChPAP may catalyze diacylglycerol pyrophosphate to phosphatidate in vacuoles.
... The use of detergent/phospholipid-mixed micelles makes it possible to analyze PAP activity that is dependent on the molar and surface concentrations of PA (3,67,68) according to the "surface dilution kinetics" model (66). The detergent/phospholipid-mixed micelle system is also useful in assessing the regulation of activity by phospholipids (69), sphingolipids (70), and nucleotides (71), as well as by the enzyme's posttranslational modification by phosphorylation (72)(73)(74)(75). ...
... Although the Triton X-100/PA-mixed micelle is useful in measuring PAP activity to assess biochemical mechanisms of the enzyme regulation (3,67,69,(71)(72)(73)(74)(75)(76), it does not simulate the membrane phospholipid bilayer. Liposomes are a widely accepted mimic of the biological membrane (77)(78)(79), and they have been used to measure PAP activity (11,65,(80)(81)(82)(83). ...
Phosphatidate phosphatase 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 and product diacylglycerol; defects in the metabolism of these lipid intermediates are the basis for lipid-based diseases such as obesity, lipodystrophy, and inflammation. The measurement of phosphatidate phosphatase 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 diacylglycerol or Pi. However, sensitivity and ease of use are variable across these methods. This review summarizes approaches to synthesize radioactive phosphatidate, to analyze radioactive and nonradioactive products diacylglycerol and Pi and discusses the advantages and disadvantages of each phosphatidate phosphatase assay.
... Studies aimed at understanding the regulation and mode of action of PA phosphatase have been subject to intense investigations (5,62). In a current working model for the regulation of yeast Pah1 PA phosphatase ( Fig. 1), the enzyme is phosphorylated in the cytoplasm by multiple protein kinases (63)(64)(65)(66)(67)(68). This posttranslational modification inhibits the PA phosphatase function by causing its retention in the cytoplasm apart from its substrate that resides in the nuclear/ER membrane (63)(64)(65)(66)69). ...
... The in vitro assay to measure yeast PA phosphatase activity has been performed with PA solubilized in the detergent Triton X-100 (1,(93)(94)(95)(96). Triton X-100 forms a uniform mixed micelle with PA, providing a surface for catalysis (93). The detergent micelle system has permitted defined studies on the kinetics of the PA phosphatase reaction (1,93) as well as on the biochemical regulation of the enzyme by phospholipids (96), sphingolipids (94), nucleotides (95), and by phosphorylation (64)(65)(66)68). Although the Triton X-100/PA-mixed micelle assay allows for defined activity measurements, it does not resemble the in vivo environment of phospholipid bilayer membrane where PA is a component. ...
In yeast and higher eukaryotes, all membrane phospholipids and the storage lipid triacylglycerol, are derived from phosphatidate (PA), which is also implicated as an activator of cell growth and proliferation, vesicular trafficking, secretion, and endocytosis. PA also regulates expression of lipid synthesis genes via the Henry (Opi1/Ino2‐Ino4) regulatory circuit, where the key step is the PA‐mediated translocation of the Opi1 repressor between the nuclear/ER membrane and the nucleus. Data indicate that several enzymes play important roles in the PA‐mediated regulation of lipid synthesis that occurs at the nuclear/ER membrane. Of the enzymes that metabolize PA, the PA phosphatase enzyme, which is encoded by the PAH1 gene, has emerged as key regulator of PA homeostasis. Indeed, cells lacking this enzyme exhibit several phenotypes that include elevated levels of PA, derepression of phospholipid synthesis genes, increased synthesis of membrane phospholipids, and the abnormal expansion of the nuclear/ER membrane. These mutant cells also exhibit a drastic reduction in TAG and lipid droplets. The PA phosphatase, which catalyzes the conversion of PA to diacylglycerol, must translocate from the cytoplasm to the ER membrane where its substrate PA resides to function in lipid synthesis. Thus, studies to examine mechanisms for the enzyme reaction at the membrane surface are warranted. In this work, we implemented defined unilamellar vesicles with a diameter of 100 nm composed of the major phospholipids within the ER membrane. Vesicles composed of phosphatidylcholine (PC) and 10 mol% PA supported PA phosphatase activity. The addition of phosphatidylethanolamine (PE) or phosphatidylinositol (PI) stimulated the activity, but phosphatidylserine (PS) had little effect. In more complex vesicle compositions, the maximum activity was observed with vesicles containing PC/PE/PS/PA or PC/PE/PI/PA. Yet, the activity was reduced when vesicles contained PC/PE/PI/PS/PA. The PA phosphatase activity was dependent on vesicle number as well as the surface concentration of PA within the vesicles. This vesicle system is being used to assess the roles of phosphorylation/dephosphorylation of Pah1 on membrane interaction and PA phosphatase activity.
Support or Funding Information
Supported by NIH grant GM028140
The Saccharomyces cerevisiae Nem1–Spo7 protein phosphatase complex dephosphorylates and thereby activates Pah1 at the nuclear/endoplasmic reticulum membrane. Pah1, a phosphatidate phosphatase catalyzing the dephosphorylation of phosphatidate to produce diacylglycerol, is one of the most highly regulated enzymes in lipid metabolism. The diacylglycerol produced in the lipid phosphatase reaction is utilized for the synthesis of triacylglycerol that is stored in lipid droplets. Disruptions of the Nem1–Spo7/Pah1 phosphatase cascade cause a plethora of physiological defects. Spo7, the regulatory subunit of the Nem1–Spo7 complex, is required for the Nem1 catalytic function and interacts with the acidic tail of Pah1. Spo7 contains three conserved homology regions (CR1–3) that are important for the interaction with Nem1, but its region for the interaction with Pah1 is unknown. Here, by deletion and site-specific mutational analyses of Spo7, we revealed that the C-terminal basic tail (residues 240-259) containing five arginine and two lysine residues is important for the Nem1–Spo7 complex–mediated dephosphorylation of Pah1 and its cellular function (triacylglycerol synthesis, lipid droplet formation, maintenance of nuclear/endoplasmic reticulum membrane morphology, and cell growth at elevated temperatures). The glutaraldehyde cross-linking analysis of synthetic peptides indicated that the Spo7 basic tail interacts with the Pah1 acidic tail. This work advances our understanding of the Spo7 function and the Nem1–Spo7/Pah1 phosphatase cascade in yeast lipid synthesis.
