Protein kinase Hsl1 phosphorylates Pah1 to inhibit phosphatidate phosphatase activity and regulate lipid synthesis in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Pah1 phosphatidate (PA) phosphatase, which catalyzes the Mg2+-dependent dephosphorylation of PA to produce diacylglycerol, plays a key role in utilizing PA for the synthesis of the neutral lipid triacylglycerol and thereby controlling the PA-derived membrane phospholipids. The enzyme function is controlled by its subcellular location as regulated by phosphorylation and dephosphorylation. Pah1 is initially inactivated in the cytosol through phosphorylation by multiple protein kinases and then activated via its recruitment and dephosphorylation by the protein phosphatase Nem1-Spo7 at the nuclear/endoplasmic reticulum membrane where the PA phosphatase reaction occurs. Many of the protein kinases that phosphorylate Pah1 have yet to be characterized with the identification of the target residues. Here, we established Pah1 as a bona fide substrate of septin-associated Hsl1, a protein kinase involved in mitotic morphogenesis checkpoint signaling. The Hsl1 activity on Pah1 was dependent on reaction time and the amounts of protein kinase, Pah1, and ATP. The Hsl1 phosphorylation of Pah1 occurred on Ser-748 and Ser-773, and the phosphorylated protein exhibited a 5-fold reduction in PA phosphatase catalytic efficiency. Analysis of cells expressing the S748A and S773A mutant forms of Pah1 indicated that Hsl1-mediated phosphorylation of Pah1 promotes membrane phospholipid synthesis at the expense of triacylglycerol, and ensures the dependence of Pah1 function on the Nem1-Spo7 protein phosphatase. This work advances understanding of how Hsl1 facilitates membrane phospholipid synthesis through the phosphorylation-mediated regulation of Pah1.

Catalytic core function of yeast Pah1 phosphatidate phosphatase reveals structural insight into its membrane localization and activity control.

The PAH1-encoded phosphatidate (PA) phosphatase is a major source of diacylglycerol for the production of the storage lipid triacylglycerol and a key regulator for the de novo phospholipid synthesis in Saccharomyces cerevisiae. The catalytic function of Pah1 depends on its membrane localization which is mediated through its phosphorylation by multiple protein kinases and dephosphorylation by the Nem1-Spo7 protein phosphatase complex. The full-length Pah1 is composed of a catalytic core (N-LIP and HAD-like domains, amphipathic helix, and the WRDPLVDID domain) and non-catalytic regulatory sequences (intrinsically disordered regions, RP domain, and acidic tail) for phosphorylation and interaction with Nem1-Spo7. How the catalytic core regulates Pah1 localization and cellular function is not clear. In this work, we analyzed a variant of Pah1 (i.e., Pah1-CC (catalytic core)) that is composed only of the catalytic core. Pah1-CC expressed on a low-copy plasmid complemented the pah1Δ mutant phenotypes (e.g., nuclear/ER membrane expansion, reduced levels of triacylglycerol, and lipid droplet formation) without requiring Nem1-Spo7. The cellular function of Pah1-CC was supported by its PA phosphatase activity mostly associated with the membrane fraction. Although functional, Pah1-CC was distinct from Pah1 in the protein and enzymological properties, which include overexpression toxicity, association with heat shock proteins, and significant reduction of the Vmax value. These findings on the Pah1 catalytic core enhance the understanding of its structural requirements for membrane localization and activity control.

The Saccharomyces cerevisiae Spo7 basic tail is required for Nem1-Spo7/Pah1 phosphatase cascade function in lipid synthesis.

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.

Phosphatidate phosphatase Pah1 contains a novel RP domain that regulates its phosphorylation and function in yeast lipid synthesis.

The Saccharomyces cerevisiae PAH1-encoded phosphatidate (PA) phosphatase, which catalyzes the Mg2+-dependent dephosphorylation of PA to produce diacylglycerol, is one of the most highly regulated enzymes in lipid metabolism. The enzyme controls whether cells utilize PA to produce membrane phospholipids or the major storage lipid triacylglycerol. PA levels, which are regulated by the enzyme reaction, also control the expression of UASINO-containing phospholipid synthesis genes via the Henry (Opi1/Ino2-Ino4) regulatory circuit. Pah1 function is largely controlled by its cellular location, which is mediated by phosphorylation and dephosphorylation. Multiple phosphorylations sequester Pah1 in the cytosol and protect it from 20S proteasome-mediated degradation. The endoplasmic reticulum-associated Nem1-Spo7 phosphatase complex recruits and dephosphorylates Pah1 allowing the enzyme to associate with and dephosphorylate its membrane-bound substrate PA. Pah1 contains domains/regions that include the N-LIP and haloacid dehalogenase-like catalytic domains, N-terminal amphipathic helix for membrane binding, C-terminal acidic tail for Nem1-Spo7 interaction, and a conserved tryptophan within the WRDPLVDID domain required for enzyme function. Through bioinformatics, molecular genetics, and biochemical approaches, we identified a novel RP (regulation of phosphorylation) domain that regulates the phosphorylation state of Pah1. We showed that the ΔRP mutation results in a 57% reduction in the endogenous phosphorylation of the enzyme (primarily at Ser-511, Ser-602, and Ser-773/Ser-774), an increase in membrane association and PA phosphatase activity, but reduced cellular abundance. This work not only identifies a novel regulatory domain within Pah1 but emphasizes the importance of the phosphorylation-based regulation of Pah1 abundance, location, and function in yeast lipid synthesis.

Characterization of an evolutionarily distinct bacterial ceramide kinase from Caulobacter crescentus.

A common feature among nearly all gram-negative bacteria is the requirement for lipopolysaccharide (LPS) in the outer leaflet of the outer membrane. LPS provides structural integrity to the bacterial membrane, which aids bacteria in maintaining their shape and acts as a barrier from environmental stress and harmful substances such as detergents and antibiotics. Recent work has demonstrated that Caulobacter crescentus can survive without LPS due to the presence of the anionic sphingolipid ceramide-phosphoglycerate (CPG). Based on genetic evidence, we predicted that protein CpgB functions as a ceramide kinase and performs the first step in generating the phosphoglycerate head group. Here, we characterized the kinase activity of recombinantly expressed CpgB and demonstrated that it can phosphorylate ceramide to form ceramide 1-phosphate. The pH optimum for CpgB was 7.5, and the enzyme required Mg2+ as a cofactor. Mn2+, but no other divalent cations, could substitute for Mg2+. Under these conditions, the enzyme exhibited typical Michaelis-Menten kinetics with respect to NBD C6-ceramide (Km,app = 19.2 ± 5.5 µM; Vmax,app = 2590 ± 230 pmol/min/mg enzyme) and ATP (Km,app = 0.29 ± 0.07 mM; Vmax,app = 10,100 ± 996 pmol/min/mg enzyme). Phylogenetic analysis of CpgB revealed that CpgB belongs to a new class of ceramide kinases, which is distinct from its eukaryotic counterpart; furthermore, the pharmacological inhibitor of human ceramide kinase (NVP-231) had no effect on CpgB. The characterization of a new bacterial ceramide kinase opens avenues for understanding the structure and function of the various microbial phosphorylated sphingolipids.

Characterization of an evolutionarily distinct bacterial ceramide kinase from Caulobacter crescentus.

A common feature among nearly all Gram-negative bacteria is the requirement for lipopolysaccharide (LPS) in the outer leaflet of the outer membrane. LPS provides structural integrity to the bacterial membrane which aids bacteria in maintaining their shape and acts as a barrier from environmental stress and harmful substances such as detergents and antibiotics. Recent work has demonstrated that Caulobacter crescentus can survive without LPS due to the presence of the anionic sphingolipid ceramide-phosphoglycerate. Based on genetic evidence, we predicted that protein CpgB functions as a ceramide kinase and performs the first step in generating the phosphoglycerate head group. Here, we characterized the kinase activity of recombinantly expressed CpgB and demonstrated that it can phosphorylate ceramide to form ceramide 1-phosphate. The pH optimum for CpgB was 7.5, and the enzyme required Mg 2+ as a cofactor. Mn 2+ , but not other divalent cations, could substitute for Mg 2+ . Under these conditions, the enzyme exhibited typical Michaelis-Menten kinetics with respect to NBD-C6-ceramide (K m,app =19.2 ± 5.5 µM; V max,app =2586.29 ± 231.99 pmol/min/mg enzyme) and ATP (K m,app =0.29 ± 0.07 mM; V max,app =10067.57 ± 996.85 pmol/min/mg enzyme). Phylogenetic analysis of CpgB revealed that CpgB belongs to a new class of ceramide kinases which is distinct from its eukaryotic counterpart; furthermore, the pharmacological inhibitor of human ceramide kinase (NVP-231) had no effect on CpgB. The characterization of a new bacterial ceramide kinase opens avenues for understanding the structure and function of the various microbial phosphorylated sphingolipids.

Conserved regions of the regulatory subunit Spo7 are required for Nem1-Spo7/Pah1 phosphatase cascade function in yeast lipid synthesis.

In the yeast Saccharomyces cerevisiae, the Nem1-Spo7 complex is a protein phosphatase that activates Pah1 phosphatidate phosphatase at the nuclear-endoplasmic reticulum membrane for the synthesis of triacylglycerol. The Nem1-Spo7/Pah1 phosphatase cascade largely controls whether phosphatidate is partitioned into the storage lipid triacylglycerol or into membrane phospholipids. The regulated synthesis of the lipids is crucial for diverse physiological processes during cell growth. Spo7 in the protein phosphatase complex is required as a regulatory subunit for the Nem1 catalytic subunit to dephosphorylate Pah1. The regulatory subunit contains three conserved homology regions (CR1, CR2, and CR3). Previous work showed that the hydrophobicity of LLI (residues 54-56) within CR1 is important for Spo7 function in the Nem1-Spo7/Pah1 phosphatase cascade. In this work, by deletion and site-specific mutational analyses, we revealed that CR2 and CR3 are also required for Spo7 function. Mutations in any one of the conserved regions were sufficient to disrupt the function of the Nem1-Spo7 complex. We determined that the uncharged hydrophilicity of STN (residues 141-143) within CR2 was required for Nem1-Spo7 complex formation. In addition, the hydrophobicity of LL (residues 217 and 219) within CR3 was important for Spo7 stability, which indirectly affected complex formation. Finally, we showed the loss of Spo7 CR2 or CR3 function by the phenotypes (e.g., reduced amounts of triacylglycerol and lipid droplets, temperature sensitivity) that are attributed to defects in membrane translocation and dephosphorylation of Pah1 by the Nem1-Spo7 complex. These findings advance knowledge of the Nem1-Spo7 complex and its role in lipid synthesis regulation.

Phosphatidic Acid Mediates the Nem1-Spo7/Pah1 Phosphatase Cascade in Yeast Lipid Synthesis.

In the yeast Saccharomyces cerevisiae, the PAH1-encoded Mg2+-dependent phosphatidate (PA) phosphatase Pah1 regulates the bifurcation of PA to diacylglycerol (DAG) for triacylglycerol (TAG) synthesis and to CDP-DAG 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. The dephosphorylated Pah1 hops onto and scoots along the membrane to recognize PA for its dephosphorylation to DAG. 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 DAG, is a significant advance to understand a regulatory cascade in yeast lipid synthesis.

The Anticancer Drug Bleomycin Shows Potent Antifungal Activity by Altering Phospholipid Biosynthesis.

Invasive fungal infections are difficult to treat with limited drug options, mainly because fungi are eukaryotes and share many cellular mechanisms with the human host. Most current antifungal drugs are either fungistatic or highly toxic. Therefore, there is a critical need to identify important fungal specific drug targets for novel antifungal development. Numerous studies have shown the fungal phosphatidylserine (PS) biosynthetic pathway to be a potential target. It is synthesized from CDP-diacylglycerol and serine, and the fungal PS synthesis route is different from that in mammalian cells, in which preexisting phospholipids are utilized to produce PS in a base-exchange reaction. In this study, we utilized a Saccharomyces cerevisiae heterologous expression system to screen for inhibitors of Cryptococcus PS synthase Cho1, a fungi-specific enzyme essential for cell viability. We identified an anticancer compound, bleomycin, as a positive candidate that showed a phospholipid-dependent antifungal effect. Its inhibition on fungal growth can be restored by ethanolamine supplementation. Further exploration of the mechanism of action showed that bleomycin treatment damaged the mitochondrial membrane in yeast cells, leading to increased generation of reactive oxygen species (ROS), whereas supplementation with ethanolamine helped to rescue bleomycin-induced damage. Our results indicate that bleomycin does not specifically inhibit the PS synthase enzyme; however, it may affect phospholipid biosynthesis through disruption of mitochondrial function, namely, the synthesis of phosphatidylethanolamine (PE) and phosphatidylcholine (PC), which helps cells maintain membrane composition and functionality. IMPORTANCE Invasive fungal pathogens cause significant morbidity and mortality, with over 1.5 million deaths annually. Because fungi are eukaryotes that share much of their cellular machinery with the host, our armamentarium of antifungal drugs is highly limited, with only three classes of antifungal drugs available. Drug toxicity and emerging resistance have limited their use. Hence, targeting fungi-specific enzymes that are important for fungal survival, growth, or virulence poses a strategy for novel antifungal development. In this study, we developed a heterologous expression system to screen for chemical compounds with activity against Cryptococcus phosphatidylserine synthase, Cho1, a fungi-specific enzyme that is essential for viability in C. neoformans. We confirmed the feasibility of this screen method and identified a previously unexplored role of the anticancer compound bleomycin in disrupting mitochondrial function and inhibiting phospholipid synthesis.

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-3ß, 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 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 (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.

Phosphorylation-mediated regulation of the Nem1-Spo7/Pah1 phosphatase cascade in yeast lipid synthesis.

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 C, 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.

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.

Lipid metabolism has been good to me.

My career in research has flourished through hard work, supportive mentors, and outstanding mentees and collaborators. The Carman laboratory has contributed to the understanding of lipid metabolism through the isolation and characterization of key lipid biosynthetic enzymes as well as through the identification of the enzyme-encoding genes. Our findings from yeast have proven to be invaluable to understand regulatory mechanisms of human lipid metabolism. Several rewarding aspects of my career have been my service to the Journal of Biological Chemistry as an editorial board member and Associate Editor, the National Institutes of Health as a member of study sections, and national and international scientific meetings as an organizer. I advise early career scientists to not assume anything, acknowledge others' accomplishments, and pay it forward.

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 Pi 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 Pi, and discusses the advantages and disadvantages of each PAP assay.

The Spo7 sequence LLI is required for Nem1-Spo7/Pah1 phosphatase cascade function in yeast lipid metabolism.

The Nem1-Spo7 complex in the yeast Saccharomyces cerevisiae is a protein phosphatase that catalyzes the dephosphory-lation of Pah1 phosphatidate phosphatase, required for its translocation to the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7/Pah1 phosphatase cascade plays a major role in triacylglycerol synthesis and in the regulation of phospholipid synthesis. In this work, we examined Spo7, a regulatory subunit required for Nem1 catalytic function, to identify residues that govern formation of the Nem1-Spo7 complex. By deletion analysis of Spo7, we identified a hydrophobic Leu-Leu-Ile (LLI) sequence comprising residues 54-56 as being required for the protein to complement the temperature-sensitive phenotype of an spo7Δ mutant strain. Mutational analysis of the LLI sequence with alanine and arginine substitutions showed that its overall hydrophobicity is crucial for the formation of the Nem1-Spo7 complex as well as for the Nem1 catalytic function on its substrate, Pah1, in vivo Consistent with the role of the Nem1-Spo7 complex in activating the function of Pah1, we found that the mutational effects of the Spo7 LLI sequence were on the Nem1-Spo7/Pah1 axis that controls lipid synthesis and related cellular processes (e.g. triacylglycerol/phospholipid synthesis, lipid droplet formation, nuclear/endoplasmic reticulum membrane morphology, vacuole fusion, and growth on glycerol medium). These findings advance the understanding of Nem1-Spo7 complex formation and its role in the phosphatase cascade that regulates the function of Pah1 phosphatidate phosphatase.

Yeast phosphatidic acid phosphatase Pah1 hops and scoots along the membrane phospholipid bilayer.

PA phosphatase, encoded by PAH1 in the yeast Saccharomyces cerevisiae, catalyzes the Mg2+-dependent dephosphorylation of PA, producing DAG at the nuclear/ER membrane. This enzyme plays a major role in triacylglycerol synthesis and in the regulation of phospholipid synthesis. As an interfacial enzyme, PA phosphatase interacts with the membrane surface, binds its substrate, and catalyzes its reaction. The Triton X-100/PA-mixed micellar system has been utilized to examine the activity and regulation of yeast PA phosphatase. This system, however, does not resemble the in vivo environment of the membrane phospholipid bilayer. We developed an assay system that mimics the nuclear/ER membrane to assess PA phosphatase activity. PA was incorporated into unilamellar phospholipid vesicles (liposomes) composed of the major nuclear/ER membrane phospholipids, PC, PE, PI, and PS. We optimized this system to support enzyme-liposome interactions and to afford activity that is greater than that obtained with the aforementioned detergent system. Activity was regulated by phospholipid composition, whereas the enzyme's interaction with liposomes was insensitive to composition. Greater activity was attained with large (≥100 nm) versus small (50 nm) vesicles. The fatty-acyl moiety of PA had no effect on this activity. PA phosphatase activity was dependent on the bulk (hopping mode) and surface (scooting mode) concentrations of PA, suggesting a mechanism by which the enzyme operates along the nuclear/ER membrane in vivo.

Phosphatidate-mediated regulation of lipid synthesis at the nuclear/endoplasmic reticulum membrane.

In yeast and higher eukaryotes, phospholipids and triacylglycerol are derived from phosphatidate at the nuclear/endoplasmic reticulum membrane. In de novo biosynthetic pathways, phosphatidate is channeled into membrane phospholipids via its conversion to CDP-diacylglycerol. Its dephosphorylation to diacylglycerol is required for the synthesis of triacylglycerol as well as for the synthesis of phosphatidylcholine and phosphatidylethanolamine via the Kennedy pathway. In addition to the role of phosphatidate as a precursor, it is a regulatory molecule in the transcriptional control of phospholipid synthesis genes via the Henry regulatory circuit. Pah1 phosphatidate phosphatase and Dgk1 diacylglycerol kinase are key players that function counteractively in the control of the phosphatidate level at the nuclear/endoplasmic reticulum membrane. Loss of Pah1 phosphatidate phosphatase activity not only affects triacylglycerol synthesis but also disturbs the balance of the phosphatidate level, resulting in the alteration of lipid synthesis and related cellular defects. The pah1Δ phenotypes requiring Dgk1 diacylglycerol kinase exemplify the importance of the phosphatidate level in the misregulation of cellular processes. The catalytic function of Pah1 requires its translocation from the cytoplasm to the nuclear/endoplasmic reticulum membrane, which is regulated through its phosphorylation in the cytoplasm by multiple protein kinases as well as through its dephosphorylation by the membrane-associated Nem1-Spo7 protein phosphatase complex. This article is part of a Special Issue entitled Endoplasmic reticulum platforms for lipid dynamics edited by Shamshad Cockcroft and Christopher Stefan.

Yck1 casein kinase I regulates the activity and phosphorylation of Pah1 phosphatidate phosphatase from Saccharomyces cerevisiae.

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 serine/threonine residues is complex and catalyzed by diverse protein kinases. In this work, we demonstrate that Pah1 is phosphorylated by the YCK1-encoded casein kinase I (CKI), regulating Pah1 catalytic activity and phosphorylation. Phosphoamino acid analysis coupled with phosphopeptide mapping of the CKI-phosphorylated Pah1 indicated that it is phosphorylated mainly on multiple serine residues. Using site-directed mutagenesis and phosphorylation analysis of Pah1, we identified eight serine residues (i.e. Ser-114, Ser-475, Ser-511, Ser-602, Ser-677, Ser-705, Ser-748, and Ser-774) as the target sites of CKI. Of these residues, Ser-475 and Ser-511 were specific for CKI, whereas the others were shared by casein kinase II (Ser-705), Cdc28-cyclin B (Ser-602), Pho85-Pho80 (Ser-114, Ser-602, and Ser-748), protein kinase A (Ser-667 and Ser-774), and protein kinase C (Ser-677). CKI-mediated phosphorylation of Pah1 stimulated both its phosphatidate phosphatase activity and its subsequent phosphorylation by casein kinase II. However, the CKI-mediated phosphorylation of Pah1 strongly inhibited its subsequent phosphorylation by Pho85-Pho80, protein kinase A, and protein kinase C. In a reciprocal analysis, Pah1 phosphorylation by Pho85-Pho80 inhibited subsequent phosphorylation by CKI. CKI-mediated Pah1 phosphorylation was also inhibited by a peptide containing the Pah1 residues 506-517, including the kinase-specific Ser-511 residue. These findings advance our understanding of how Pah1 catalytic activity and phosphorylation are regulated by multiple protein kinases.

Protein kinase C mediates the phosphorylation of the Nem1-Spo7 protein phosphatase complex in yeast.

The Nem1-Spo7 complex in the yeast Saccharomyces cerevisiae is a protein phosphatase required for the nuclear/endoplasmic reticulum membrane localization of Pah1, a phosphatidate phosphatase that produces diacylglycerol for triacylglycerol synthesis at the expense of phospholipid synthesis. In a previous study, we showed that the protein phosphatase is subject to phosphorylation by protein kinase A (PKA). Here, we demonstrate that Nem1-Spo7 is regulated through its phosphorylation by protein kinase C (PKC), which plays multiple roles, including the regulation of lipid synthesis and cell wall integrity. Phosphorylation analyses of Nem1-Spo7 and its synthetic peptides indicate that both subunits of the complex are bona fide PKC substrates. Site-directed mutagenesis of NEM1 and SPO7, coupled with phosphopeptide mapping and immunoblotting with a phosphoserine-specific PKC substrate antibody, revealed that Ser-201 in Nem1 and Ser-22/Ser-28 in Spo7 are major PKC target sites of phosphorylation. Activity analysis of mutant Nem1-Spo7 complexes indicates that the PKC phosphorylation of Nem1 exerts a stimulatory effect, but the phosphorylation of Spo7 has no effect. Lipid-labeling analysis of cells expressing the phosphorylation-deficient alleles of NEM1 and SPO7 indicates that the stimulation of the Nem1-Spo7 activity has the effect of increasing triacylglycerol synthesis. Prephosphorylation of Nem1-Spo7 by PKC inhibits the PKA phosphorylation of Nem1, whereas prephosphorylation of the phosphatase complex by PKA inhibits the PKC phosphorylation of Spo7. Collectively, this work advances the understanding of the Nem1-Spo7 regulation by phosphorylation and its impact on lipid synthesis.

Phosphatidylserine synthesis is essential for viability of the human fungal pathogen Cryptococcus neoformans.

Phospholipids are an integral part of the cellular membrane structure and can be produced by a de novo biosynthetic pathway and, alternatively, by the Kennedy pathway. Studies in several yeast species have shown that the phospholipid phosphatidylserine (PS) is synthesized from CDP-diacylglycerol and serine, a route that is different from its synthesis in mammalian cells, involving a base-exchange reaction from preexisting phospholipids. Fungal-specific PS synthesis has been shown to play an important role in fungal virulence and has been proposed as an attractive drug target. However, PS synthase, which catalyzes this reaction, has not been studied in the human fungal pathogen Cryptococcus neoformans Here, we identified and characterized the PS synthase homolog (Cn Cho1) in this fungus. Heterologous expression of Cn CHO1 in a Saccharomyces cerevisiae cho1Δ mutant rescued the mutant's growth defect in the absence of ethanolamine supplementation. Moreover, an Sc cho1Δ mutant expressing Cn CHO1 had PS synthase activity, confirming that the Cn CHO1 encodes PS synthase. We also found that PS synthase in C. neoformans is localized to the endoplasmic reticulum and that it is essential for mitochondrial function and cell viability. Of note, its deficiency could not be complemented by ethanolamine or choline supplementation for the synthesis of phosphatidylethanolamine (PE) or phosphatidylcholine (PC) via the Kennedy pathway. These findings improve our understanding of phospholipid synthesis in a pathogenic fungus and indicate that PS synthase may be a useful target for antifungal drugs.