FIGURE 3 - uploaded by George M Carman
Content may be subject to copyright.
Characterization of PKC activity using Pah1p as a substrate. Phosphorylation of Pah1p by PKC was measured by following the incorporation of the radiolabeled phosphate from [-32 P]ATP into purified recombinant Pah1p under standard reaction conditions by varying the time (A), amount of PKC (B), and concentrations of ATP (C) and Pah1p (D). Following the reactions, the samples were subjected to SDS-PAGE; the polyacrylamide gels were dried and then subjected to phosphorimaging analysis. The relative amounts of phosphate incorporated into Pah1p were quantified using ImageQuant software. The data shown in A-D are the averages of three experiments S.D. (error bars).
Source publication
Yeast Pah1p is the phosphatidate phosphatase that catalyzes the penultimate step in triacylglycerol synthesis and plays a role in the transcriptional regulation of phospholipid synthesis genes. The enzyme is multiply phosphorylated, some of which is mediated by Pho85p-Pho80p, Cdc28p-cyclin B, and protein kinase A. Here, we showed that Pah1p is a bo...
Contexts in source publication
Context 1
... of the radioactive phosphate from [-32 P]ATP into Pah1p. Phosphorylated Pah1p was resolved by SDS-PAGE and then detected by phosphorim- aging ( Fig. 2A). Phosphoamino acid analysis showed that Pah1p is phosphorylated by PKC on the serine residue (Fig. 2B). The Pah1p phosphorylation was dependent on the reaction time and the amount of PKC (Fig. 3, A and B, respectively), indicat-ing that it is a bona fide substrate of PKC. In addition, PKC activity followed Michaelis-Menten kinetics with respect to ATP and Pah1p (Fig. 3, C and D, respectively). The K m for Pah1p was 0.75 M, and this value is higher than that shown by PKA, Pho85p-Pho80p, or Cdc28p-cyclin B (Table 2). At the point of maximum ...
Context 2
... analysis showed that Pah1p is phosphorylated by PKC on the serine residue (Fig. 2B). The Pah1p phosphorylation was dependent on the reaction time and the amount of PKC (Fig. 3, A and B, respectively), indicat-ing that it is a bona fide substrate of PKC. In addition, PKC activity followed Michaelis-Menten kinetics with respect to ATP and Pah1p (Fig. 3, C and D, respectively). The K m for Pah1p was 0.75 M, and this value is higher than that shown by PKA, Pho85p-Pho80p, or Cdc28p-cyclin B (Table 2). At the point of maximum phosphorylation, PKC catalyzed the incorporation of 0.8 mol of phosphate/mol of ...
Context 3
... that Ser-677 and Ser-769 are additional phosphorylation sites. By comparing the phosphopeptide maps of S677A, S769A, S773A, and S788A mutants with that of the wild type Pah1p, we could ascribe which site was contained within the phosphopeptide map (Fig. 5D, WT). Multiple phosphopeptides ascribed to phosphory- lated Ser-677 and Ser-769 ( Fig. 3D, S677A and S769A) are thought to result from incomplete proteolytic digestion at sites that are in close proximity to the phosphorylated serine resi- dues. Overall, four serine residues (Ser-677, Ser-769, Ser-773, and Ser-788) at the C-terminal region of Pah1p were identified as major sites of phosphorylation by ...
Context 4
... effects of the Pah1p phosphorylation by PKC were sig- nificantly different from those of Pho85p-Pho80p. The phos- phorylation by Pho85p-Pho80p prevents Pah1p from associat- ing with the nuclear/endoplasmic reticulum membrane and inhibits PAP activity and the synthesis of TAG (36, 37, 80). The indicated wild type and phosphorylation site mutant forms of Pah1p were expressed in pah1 and pah1 nem1 cells. ...
Similar publications
The Nem1-Spo7 protein phosphatase plays a role in lipid synthesis by controlling the membrane localization of Pah1, the diacylglycerol-producing phosphatidate (PA) phosphatase that is crucial for the synthesis of triacylglycerol in the yeast Saccharomyces cerevisiae By dephosphorylating Pah1, Nem1-Spo7 facilitates its translocation to the nuclear/e...
Pah1 phosphatidate phosphatase inSaccharomyces cerevisiaecatalyzes the penultimate step in the synthesis of triacylglycerol, i.e., the production of diacylglycerol by dephosphorylation of phosphatidate. The enzyme playing a major role in lipid metabolism is subject to phosphorylation (e.g., by Pho85-Pho80, Cdc28-cyclin B, protein kinases A and C) a...
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...
Citations
... Since the TORC2-Ypk1/2 signaling controls the synthesis of LCBs and ceramides through regulating serine palmitoyltransferase and ceramide synthase 18-21 , our findings suggest that the CWI pathway negatively regulates sphingolipid biosynthesis by affecting multiple steps during ceramide synthesis through different mechanisms. Pkc1 has also been reported to target some proteins involved in phospholipid metabolism, including Pah1 (phosphatidate phosphatase) 73 , Ura7 (CTP synthase) 74 , and Opi1 (transcriptional repressor) 75 . The CWI pathway may control phospholipid metabolism in addition to sphingolipid metabolism through Pkc1 signaling, and this control of lipid metabolisms may contribute to the adaptive response to cell wall stress. ...
The target of rapamycin complex 2 (TORC2) signaling is associated with plasma membrane (PM) integrity. In Saccharomyces cerevisiae, TORC2-Ypk1/2 signaling controls sphingolipid biosynthesis, and Ypk1/2 phosphorylation by TORC2 under PM stress conditions is increased in a Slm1/2-dependent manner, under which Slm1 is known to be released from an eisosome, a furrow-like invagination PM structure. However, it remains unsolved how the activation machinery of TORC2-Ypk1/2 signaling is regulated. Here we show that edelfosine, a synthetic lysophospholipid analog, inhibits the activation of TORC2-Ypk1/2 signaling, and the cell wall integrity (CWI) pathway is involved in this inhibitory effect. The activation of CWI pathway blocked the eisosome disassembly promoted by PM stress and the release of Slm1 from eisosomes. Constitutive activation of TORC2-Ypk1/2 signaling exhibited increased sensitivity to cell wall stress. We propose that the CWI pathway negatively regulates the TORC2-Ypk1/2 signaling, which is involved in the regulatory mechanism to ensure the proper stress response to cell wall damage.
... In yeasts, the function of Nem1 in lipid metabolism has been widely studied (12)(13)(14). As the catalytic subunit, Nem1 forms a phosphatase holoenzyme with its regulatory subunit Spo7 (sporulation-specific protein 7), and the Nem1/Spo7 complex is involved in regulating nuclear/ endoplasmic reticulum (ER) membrane association of Pah1, a phosphatidic acid (PA) phosphatase (15)(16)(17)(18)(19). Pah1 is phosphorylated by multiple kinases and dephosphorylated by Nem1/Spo7 complex (20)(21)(22)(23)(24); the dephosphorylated Pah1 is active and then dephosphorylates PA to produce diacylglycerol (DAG), which is the precursor of phospholipids through the de novo (Kennedy) pathway and also is required for the formation of triacylglycerols (TAGs), the most prominent neutral lipids in lipid droplets (LDs) (12,25). In addition, disruption of yeast Nem1/Spo7 complex results in defects in sporulation, premeiotic replication, and autophagy (19,26,27). ...
Protein phosphatase complex Nem1/Spo7 plays crucial roles in the regulation of various biological processes in eukaryotes. However, its biological functions in phytopathogenic fungi are not well understood. In this study, genome-wide transcriptional profiling analysis revealed that Nem1 was significantly upregulated during the infection process of Botryosphaeria dothidea, and we identified and characterized the phosphatase complex Nem1/Spo7 and its substrate Pah1 (a phosphatidic acid phosphatase) in B. dothidea. Nem1/Spo7 physically interacted with and dephosphorylated Pah1 to promote triacylglycerol (TAG) and subsequent lipid droplet (LD) synthesis. Moreover, the Nem1/Spo7-dependently dephosphorylated Pah1 functioned as a transcriptional repressor of the key nuclear membrane biosynthesis genes to regulate nuclear membrane morphology. In addition, phenotypic analyses showed that the phosphatase cascade Nem1/Spo7-Pah1 was involved in regulating mycelial growth, asexual development, stress responses, and virulence of B. dothidea.
IMPORTANCE Botryosphaeria canker and fruit rot caused by the fungus Botryosphaeria dothidea is one of the most destructive diseases of apple worldwide. Our data indicated that the phosphatase cascade Nem1/Spo7-Pah1 plays important roles in the regulation of fungal growth, development, lipid homeostasis, environmental stress responses, and virulence in B. dothidea. The findings will contribute to the in-depth and comprehensive understanding of Nem1/Spo7-Pah1 in fungi and the development of target-based fungicides for disease management.
... 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 is easily degraded by the 20S proteasome when it is dephosphorylated (or unphosphorylated) but stable against the proteasomal degradation when it is phosphorylated (63,64). An exception to the protective effect of phosphorylation is shown by protein kinase C whose activity on Pah1 lacking prephosphorylation by Pho85-Pho80 stimulates its proteasomal degradation (50). The Nem1 and Spo7 subunits, which form a phosphatase complex to regulate the phosphorylation state of Pah1, are themselves subject to phosphorylation (44,(65)(66)(67)(68), adding additional control of Pah1 function by phosphorylation. ...
... When compared as a substrate, Pah1 phosphorylated by Rim11 is not as good as that phosphorylated by Pho85-Pho80 but better than that phosphorylated by other protein kinases. Of the known protein kinase-phosphorylation site relationships in Pah1 (47)(48)(49)(50)(51), the activity of the Nem1-Spo7 protein phosphatase is in the order of the sites phosphorylated by Pho85-Pho80 (100%) > Rim11 (38%) > protein kinase A (25%) = CKII (25%) > Cdc28-cyclin B (15%) > PKC (7.5%) (51). ...
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. ...
... The dephosphorylated (or unphosphorylated) form of Pah1 is susceptible to degradation by the 20S proteasome, whereas the phosphorylated form is stable against the proteasomal degradation (Hsieh et al., 2015;Pascual et al., 2014). An exception to the phosphorylation effect is shown by protein kinase C (PKC), which stimulates the proteasomal degradation of Pah1 in the absence of prephosphorylation by the Pho85-Pho80 protein kinase, (Su et al., 2014a). Whereas the Nem1-Spo7 complex functions to dephosphorylate Pah1, both Nem1 and Spo7 are themselves subject to phosphorylation (Dey et al., 2017;Dubots et al., 2014;Holt et al., 2009;Su et al., 2018;Swaney et al., 2013), and these modifications add yet another layer of complexity to the regulation of Pah1 PAP function. ...
... Pah1 is phosphorylated to control its subcellular location, catalytic activity, and protein stability , and its phosphorylation sites are concentrated in the intrinsically disordered regions (IDRs) ( Fig. 2A) located between the two conserved domains and at the C-terminal region (Hsieh et al., 2015). Many of those sites have been identified as target residues for specific protein kinases (e.g., casein kinase I (CKI) , casein kinase II (CKII) (Hsieh et al., 2016), Cdc28-cyclin B , Pho85-Pho80 (Choi et al., 2012), protein kinase A (PKA) , and PKC (Su et al., 2014a)). ...
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 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). ...
... 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). Phosphorylated PA phosphatase is then recruited to the membrane through its association and dephosphorylation by the Nem1-Spo7 protein phosphatase complex (32,41,70). ...
... 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
... Budding yeast has only a single Pah1 ( Han et al., 2006), whereas mammalian cells contain five homologous proteins, lipin-1a, -1b, and -1c and lipin-2 and lipin-3, with different tissue distributions ( Peterfy et al., 2001;Donkor et al., 2007). Phosphatidate phosphatase (PAP) activity of Pah1 is largely dependent on its phosphorylation status, which is regulated by multiple protein kinases, including Pho85-Pho80 ( Choi et al., 2012), Cdc28-cyclin B (Choi et al., 2011), PKA ( Su et al., 2012) and PKC ( Su et al., 2014). Simultaneously, the Nem1-Spo7 protein phosphatase complex dephosphorylates Pah1 at the nuclear/endoplasmic reticulum membrane, thereby increasing the catalytic activity of Pah1 and reducing the protein stability (O' Hara et al., 2006;Pascual et al., 2014). ...
... Mammalian cells contain five homologous proteins encoded by three lipin genes (LIPIN 1, LIPIN 2, and LIPIN 3) that are crucial regulators of lipid homeostasis. Budding yeast bears a single lipin, Pah1, which has been identified to be a target of multiple protein kinases, including Pho85-Pho80 ( Choi et al., 2012), Cdc28-cyclin B (Choi et al., 2011), PKA ( Su et al., 2012) and PKC ( Su et al., 2014). Moreover, mutations in seven Ser/Thr sites (S110A/ S114A/S168A/S602A/T723A/S744A/S748A) phosphorylated by Pho85-Pho80 and/or Cdc28-cyclin B completely abolished phosphorylation of Pah1, and led to its dysfunction (O' Hara et al., 2006). ...
Lipid droplets (LDs) control lipid metabolism in eukaryotic cells in general. However, the biogenesis regulation and biological functions of LDs are largely unknown in pathogenic fungi.
Rapamycin treatment results in a significant increase of LD biogenesis in Fusarium graminearum. Molecular mechanisms of the target of rapamycin (TOR) pathway in regulating LD biogenesis and the functions of LD in virulence of F. graminearum were investigated in depth by combining genetic, cytological and phenotypic strategies.
TOR in Fusarium graminearum (FgTOR) inhibition by rapamycin induces LD biogenesis through the FgPpg1/Sit4 signaling branch. FgPpg1 promotes phosphorylation of protein phosphatase FgNem1 by the protein kinase FgCak1. The phosphorylated FgNem1 dephosphorylates the phosphatidate phosphatase FgPah1. Dephosphorylated FgPah1 is active and stimulates LD biogenesis. Moreover, deletion of FgNem1/Spo7 or FgPah1 leads to serious defects in vegetative growth, sexual development and virulence.
The results of this study provide novel insights into the regulatory mechanism and biological functions of the LDs in the devastating pathogenic fungus F. graminearum.
